Invivo agents comprising cationic metal chelators with acidic saccharides and glycosaminoglycans

This application concerns novel agents comprising cationic or chemically basic metal chelators in association with hydrophilic carriers of anionic or chemically acidic saccharides, sulfatoids and glycosaminoglycans. In certain embodiments, the agents comprise metals and metal ions. Covalent and non-covalent chemical and physical means are described for stabilizing the binding of the metal chelators to the carriers. Novel non-covalently bound compositions are described which give uniquely high payloads and ratio of metal chelator to carrier, ranging from a low of about 15% metal chelator by weight, to a characteristic range of 70% to 90% metal chelator by weight. Specific embodiments are described comprising deferoxamine, ferrioxamine, iron-basic porphine, iron-triethylenetetraamine, gadolinium DTPA-lysine, gadolinium DOTA-lysine and gadolinium with basic derivatives of porphyrins, porphines, expanded porphyrins, Texaphyrins and sapphyrins as the basic or cationic metal chelators, which are in turn, bound to acidic or anionic carriers, including one or more of acidic or anionic saccharides, and including sulfated sucrose, pentosan polysulfate, dermatan sulfate, oversulfated dermatan sulfate, chondroitin sulfate, oversulfated chondroitin sulfate, heparan sulfate, beef heparin, porcine heparin, non-anticoagulant heparins, and other native and modified acidic saccharides and glycosaminoglycans. Also disclosed are methods of enhancing in vivo imgages arising from induced magnetic resonance signals, methods of enhancing in vivo images in conjunction with ultrasound or X-rays and methods of obtaining in vivo body images utilizing radioisotope containing agents. Methods of treating vasular disease are also disclosed.

The present invention describes novel compositions, agents and methods of 
in vivo use which give improved selectivity, efficacy, uptake mechanism 
and kinetic-spatial profiles at sites of disease. It further describes 
compositions, agents and methods of use for improved selectivity, 
sensitivity, uptake mechanism and kinetic-spatial profiles of biomedical 
imaging, image contrast and spectral enhancement at sites of disease, 
including but not limited to magnetic resonance image (MRI) contrast 
enhancement. Novel compositions are prepared by (a) unique non-covalent 
chemical binding, further enhanced by (b) physical stabilization. Other 
compositions are prepared by covalent chemical binding. Binding is of 
cationic or chemically basic metal chelators to carriers comprising 
anionic or chemically acidic saccharides, sulfatoids and 
glycosaminoglycans, typically and advantageously of a hydrophilic or 
essentially completely hydrophilic nature. Binding of the active and 
carrier may also be by a combination of non-covalent, physical, and 
covalent means. Non-covalent binding can be carried out by means including 
but not limited to admixing cationic or basic metal chelators at 
appropriate ratios with anionic or acidic saccharide carriers, thereby 
forming solution-state and dry-state paired-ion salts, based principally 
on electrostatic binding of cationic (basic) group or groups of the metal 
chelator to anionic (acidic) group or groups of the acidic carrier. Such 
binding may be further stabilized by hydrogen bonds and physical factors, 
including but not limited to concentration, viscosity, and various means 
of drying, including lyophilization. 
Carrier substances useful in this invention may include, but are not 
limited to natural and synthetic, native and modified, anionic or acidic 
saccharides, disaccharides, oligosaccharides, polysaccharides and 
glycosaminoglycans (GAGs). It will be apparent to those skilled in the art 
that a wide variety of additional biologically compatible, water-soluble 
and water dispersable, anionic carrier substances can also be used. Due to 
an absence of water-diffusion barriers, favorable initial biodistribution 
and multivalent site-binding properties, oligomeric and polymeric, 
hydrophilic and substantially completely hydrophilic carrier substances 
are included among the preferred carriers for agents to be used for 
paramagnetic, T1-Type, selective MRI contrast of tumors, cardiovascular 
infarcts and other T1-Type MRI contrast uses. However, it will be apparent 
to those skilled in the art that amphoteric and hydrophobic carriers may 
be favored for certain biomedical imaging applications and therapeutic 
applications. Metal chelators useful in this invention include those which 
contain cationic, basic and basic-amine groups and which chelate metals 
and metal ions, transition elements and ions, and lanthanide series 
elements and ions. It will be apparent to those skilled in the art that 
essentially any single atomic element or ion amenable to chelation by a 
cationic, basic and amine-containing chelator, may also be useful in this 
invention. 
For purposes of this invention, a cationic or basic metal chelator is 
defined and further distinguished from a metal-ion complex as follows: a 
cationic or basic metal chelator comprises an organic, covalent, 
bridge-ligand molecule, capable of partly or entirely surrounding a single 
metal atom or ion, wherein the resulting formation constant of chelator 
for appropriate metal or ion is at least about 10.sup.14. A chelator is 
further defined as cationic or basic if it or its functional group or 
groups which confer the cationic or basic property, and which include but 
are not limited to an amine or amines, is (are) completely or essentially 
completely electrophilic, positively charged or protonated at a typical pH 
employed for formulation. A formulation pH is characteristically selected 
to closely bracket the range of physiologic pH present in mammalian 
vertebrates. This typically includes, but is not limited to a pH in the 
range of pH 5 to 8. Amines may include primary, secondary, tertiary or 
quaternary amines or combinations thereof on the metal chelator. Herein, 
and as specified, a hydrophilic carrier is defined as a substance which is 
water soluble, partitions into the water phase of aqueous-organic solvent 
mixtures, or forms a translucent aqueous solution, complex, aggregate, or 
particulate dispersion under the conditions employed for formulation. A 
carrier is further defined as being anionic or acidic if it is completely 
or nearly completely nucleophilic, or if its functional group or groups 
capable of interacting with cationic, basic or amine metal chelators, is 
(are) completely or nearly completely negatively charged, anionic or 
ionized at the pH employed for formulation. Such anionic and acidic groups 
include, but are not limited to sulfates, phosphates and carboxylates, or 
combinations thereof on the carrier. 
Novel agent compositions include, but are not limited to the classes of 
cationic or basic, typically basic-amine metal chelator actives, or metal 
chelator actives including the chelated metal or metal ion, wherein these 
actives are further bound to anionic and acidic carriers comprising 
natural, synthetic or synthetic carriers, including but not limited to 
hydrophilic anionic or acidic, natural or synthetic, native, modified, 
derivatized and fragmented, anionic or acidic saccharides, 
oligosaccharides, polysaccharides, sulfatoids, and glycosaminoglycans 
(GAGs). 
Anionic and acidic saccharide and glycosaminoglycan carriers may contain 
monomeric units comprising glucose, glucuronic acid, iduronic acid, 
glucosamine, galactose, galactosamine, xylose, mannose, fucose, sialic 
acid, pentose, and other naturally occurring, semi-synthetic or synthetic 
monosaccharides or chemical derivatives thereof, comprising amine, 
sulfate, carboxylate, sialyl, phosphate, hydroxyl or other side groups. 
Glycosaminoglycans (GAGs) comprise essentially the carbohydrate portions 
of cell-surface and tissue matrix proteoglycans. They are derived from 
naturally occurring proteoglycans by chemical separation and extraction; 
and in certain instances, by enzymatic means Lindahl et al. (1978), 
incorporated herein by reference!. They include, but are not limited to 
those of the following types: heparin, heparan sulfate, dermatan sulfate, 
chondroitin-4-sulfate, chondroitin-6-sulfate, keratan sulfate, syndecan, 
and hyaluronate, and over-sulfated, hyper-sulfated, and other chemical 
derivatives thereof, as described further below. 
Strongly acidic glycosaminoglycans include all of those classes listed just 
above, except for hyaluronate, which contains only the more weakly acidic 
carboxylate groups and not sulfate groups. Natural sources of 
glycosaminoglycans include, but are not limited to: pig and beef 
intestinal mucosa, lung, spleen, pancreas, and a variety of other solid 
and parenchymal organs and tissues. 
Sulfatoids comprise a second class of sulfated saccharide substances which 
are derived principally but not exclusively from bacterial and 
non-mammalian sources. Sulfatoids are typically of shorter chain length 
and lower molecular weight than glycosaminoglycans, but may be 
synthetically modified to give (a) longer chain lengths, (b) increased 
sulfation per unit saccharide, (c) various other chemical side groups, or 
(c) other properties favorable to the desired ligand-binding property and 
site-selective binding, uptake and accumulation property (or properties) 
in vivo. Sucrose and other short-chain oligosaccharides may be obtained 
from natural and synthetic sources. 
These oligosaccharides can be rendered anionic or acidic by chemical or 
enzymatic derivatization with carboxylate, phosphate, sulfate or silyl 
side groups, or combinations thereof, at substitution ratios of up to 
about eight anionic or acidic substituent groups per disaccharide unit. 
Modified glycosaminoglycans may be derived from any of the types and 
sources of native glycosaminoglycans described above, and include: (1) 
glycosaminoglycan fragments, further defined as glycosaminoglycans with 
chain lengths made shorter than the parental material as isolated directly 
from natural sources by standard ion-exchange separation and solvent 
fractionation methods; (2) glycosaminoglycans chemically modified to 
decrease their anticoagulant activities, thereby giving 
"non-anticoagulant" (NAC) GAGs, prepared typically but not exclusively by 
(a) periodate oxidation followed by borohydride reduction; (b) partial or 
complete desulfation; and (c) formation of non-covalent divalent or 
trivalent counterion salts, principally including but not limited to salts 
of the more highly acidic sulfate functional groups, with principally but 
not exclusively: calcium, magnesium, manganese, iron, gadolinium and 
aluminum ions. 
For purposes of this invention, a special class of such salts includes 
those salts formed by electrostatic or paired-ion association between the 
acidic or sulfate groups of acidic saccharide or glycosaminoglycan 
carrier, and the basic or cationic group or groups of the metal chelator 
or metal chelator including metal, as described above. Derivatized acidic 
saccharides and glycosaminoglycans are typically prepared by 
derivatization of various chemical side groups to various sites on the 
saccharide units. This may be performed by chemical or enzymatic means. 
Enzymatic means are used in certain instances where highly selective 
derivatization is desired. Resulting chemical and enzymatic derivatives 
include, but are not limited to acidic saccharides and glycosaminoglycans 
derivatized by: (1) esterification of (a) carboxylate groups, (b) hydroxyl 
groups, and (c) sulfate groups; (2) oversulfation by nonselective chemical 
or selective enzymatic means; (3) acetylation, and (4) formation of 
various other ligand derivatives, including but not limited to (a) 
addition of sialyl side groups, (b) addition of fucosyl side groups, and 
(c) treatment with various carbodiimide, anhydride and isothiocyanate 
linking groups, and (d) addition of various other ligands. 
If and when present, sulfate and sialyl side groups may be present at any 
compatible position of saccharide monomer, and on any compatible position 
of glycosaminoglycan monomers Lindahl et al. (1978), incorporated herein 
by reference!. Certain of the resulting derivatized acidic saccharides and 
glycosaminoglycans may have desired alterations of anticoagulant 
activities, site-localization patterns, clearance and other biological 
properties. As one example of this relationship between certain classes of 
glycosaminoglycans and biological properties, dermatan sulfates with a 
native sulfate/carboxylate ratio of 1:1, are reported to have relatively 
low binding to normal endothelial cells, avoid displacement of endogenous 
heparan sulfate from endothelial-cell surfaces, have relatively high 
selectivity to induced endothelia at sites of disease, including thrombus, 
and have rapid plasma clearance, principally by the renal route; whereas 
heparins and oversulfated dermatan sulfates with higher 
sulfate/carboxylate ratios of between 2:1 and 3.7:1, are reported to have 
relatively higher binding for both normal and induced endothelia, to 
displace relatively more endogenous endothelial heparan sulfate, and to 
clear more slowly than dermatans Boneu et al. (1992), incorporated herein 
by reference!. 
In a special case unique to the present invention, derivatization of the 
acidic saccharide and glycosaminoglycan carriers may be accompanied by the 
basic metal chelator itself. Although the general classes of carriers 
described above are particularly suitable to the present invention, it 
will be apparent to those skilled in the art that a wide variety of 
additional native, derivatized and otherwise modified carriers and 
physical formulations thereof, may be particularly suitable for various 
applications of this invention. As one representative example, the source 
and type of glycosaminoglycans, its chain length and sulfate/carboxylate 
ratio can be optimized to (1) provide optimal formulation characteristics 
in combination with different small and macromolecular diagnostic agents 
and drugs; (2) modulate carrier localization on diseased versus normal 
endothelium; (3) minimize dose-related side effects; (4) optimize 
clearance rates and routes of the carrier and bound diagnostic and 
therapeutic actives. 
Non-covalent formulations of active and carrier afford markedly higher 
active-to-carrier ratios than those possible for covalent chemical 
conjugates. In the present invention, non-covalent binding affords a 
minimum of 15% active to total agent by weight active/(active+carrier), 
w/w!; typically greater than about 30% (w/w); preferably at least about 
50% (w/w); and frequently between about 70-99% (w/w). Covalent binding 
characteristically limits the percent active to (a) less than about 12% 
for non-protein small and polymeric carriers, (b) less than about 7% for 
peptide and protein carriers, including antibodies, and (c) less than 
about 0.5-2.0% for antibody fragments. This limitation is based on the 
number of functional groups available on carrier molecules which are 
useful in agent formulation and in vivo site localization. 
It will be apparent to those skilled in the art that covalent 
active-carrier agent compositions of low substitution ratio may be useful 
for certain in vivo applications of typically narrow range, and that 
non-covalent active-carrier agent compositions of high substitution ratio 
may be useful for other in vivo applications of typically broader range. 
Generally, but not exclusively, covalent agents may be useful for 
radionuclide imaging or therapeutic applications in which only low 
total-body doses are needed, clearance of the non-targeted dose fraction 
does not cause undue toxicity, and high conjugate stability is required. 
Generally, but not exclusively, non-covalent agents may be particularly 
useful for the majority of diagnostic imaging applications and certain 
high-dose therapeutic applications, for which high total-body and 
site-localized doses are needed, and rapid clearance of the non-localized 
fraction of administered agent is desired in order to accelerate plasma 
clearance and to achieve low background levels for purposes of maximizing 
image contrast and minimizing systemic toxicity. 
Rapid clearance is preferentially conferred by non-covalent physical 
formulations due to their capacity to give controlled dissociation or 
release of the active from the carrier. Such controlled release allows the 
diagnostic or therapeutic active, to dissociate from its carrier at a 
programmed rate which is consistent with rapid site localization of a 
significant fraction of the total administered dose. In instances where 
the carrier is polymeric and hence clears more slowly, this selectively 
accelerates clearance of the active. 
It will be apparent to those skilled in the art that such controlled 
release can also be achieved for actives which are chemically conjugated 
to their carriers via chemical linkers, including peptide linkers, which 
are susceptible to cleavage by body enzymes. However, this latter means of 
facilitated clearance: (a) gives much longer clearance times than do 
physical formulations, (b) depends on endogenous enzyme levels and 
inhibitors which typically differ from subject to subject, from health to 
disease, and from one stage of disease to another. Hence, physical 
formulations have substantial advantages over chemical conjugates from the 
standpoints of both (a) high payload, and (b) accelerated clearance. 
These properties of the present formulations represent additional 
substantial improvements over prior, non-selective and covalently 
conjugated active-carrier agents. The resulting agents are broadly useful 
for: (a) MRI contrast and spectral enhancement, Ultrasound contrast 
enhancement, and X-Ray contrast enhancement, where relatively high 
administered doses may be favored or required; (b) Nuclear Medical or 
Radionuclide imaging and therapy, where enhanced clearance of the 
non-targeted dose may be favored or required: and (c) certain high-dose, 
extended-release or sustained-effect therapy may be favored or required. 
Such therapeutic agents include but are not limited to those useful at a 
broad variety of organ sites and medical indications, for the treatment 
of: (a) acute vascular ischemia, acute infarct, acute vascular damage, 
shock, hypotension, restenosis, proliferation of neo-vessel, parenchymal 
cells or other pathological proliferations; and (b) the following classes 
of disease: vascular, parenchymal, mesenchymal, endothelial, smooth 
muscle, striated muscle, adventitial, immune, inflammatory, bacterial, 
fungal, viral, degenerative, neoplastic, genetic and enzymatic. 
MRI contrast enhancement is one important indication for which high payload 
and controlled release of active are important unique advantages in 
addition to site selective localization (see below). A still further 
advantage is the hydrophilic form of carrier, which maximizes proximal 
water diffusion and binding of the paramagnetic active. This last property 
is required for optimal efficacy and minimal toxicity, because MRI 
paramagnetic T1-Type contrast agents require unimpeded water diffusion to 
within a very short distance of the localized metal ion in order to 
achieve effective paramagnetic relaxation and T1 contrast. Additionally, 
MRI image instrumentation and image acquisition are inherently both of low 
sensitivity; and these limitations remain even at the highest clinically 
acceptable field strengths and gradients and at the optimal radiofrequency 
pulse sequences. 
MRI paramagnetic agents have been prepared as stabilized liposomes, which 
contain up to about 22% of active (w/w). However, their hydrophobic lipid 
bilayers markedly impede water diffusion into the liposome core active. 
This decreases their efficacy per unit dose relative to the hydrophilic 
controlled-release carriers of the present invention. There is an 
additional disadvantage of the reported MRI liposome formulations as 
follows: aside from localization in normal liver and 
reticuloendothelial-phagocytic organs, they have not demonstrated 
effective site-localization at sites of tumors, infarcts and other focal 
pathology within tissue sites. 
For purposes of this invention, metal ions generally useful for chelation 
in paramagnetic T1-Type MRI contrast agent compositions and uses may 
include divalent and trivalent cations selected from the group consisting 
of: iron, manganese, chromium, copper, nickel, gadolinium, erbium, 
europium, dysprosium and holmium. Chelated metal ions generally useful for 
radionuclide imaging and compositions and uses, and in radiotherapeutic 
compositions and uses, may include metals selected from the group of: 
gallium, germanium, cobalt, calcium, rubidium, yttrium, technetium, 
ruthenium, rhenium, indium, iridium, platinum, thallium and samarium. 
Metal ions useful in neutron-capture radiation therapy may include boron 
and others with large nuclear cross sections. Metal ions useful in 
Ultrasound contrast and X-Ray contrast compositions and uses may, provided 
they achieve adequate site concentrations, include any of the metal ions 
listed above, and in particular, may include metal ions of atomic number 
at least equal to that of iron. 
For purposes of this invention, agents for therapeutic composition and uses 
in chelating internal body iron, copper or both, in order to make these 
metals unavailable locally (1) which are typically required for 
neovascularization, or (2) which cause and amplify local tissue injury 
Levine (1993), incorporated herein by reference!, include the carrier 
with basic metal chelator in one or both of the following forms: (a) 
carrier plus chelator without metal ion; and (b) carrier plus chelator 
with metal ion added and chelated in the composition at a formation 
constant lower or equal to that of the internal body metal which is to be 
chelated by metal ion exchange into the respective basic metal chelator of 
the composition (see below). Such weakly chelated metal ions of the 
composition may include one selected from the group of: calcium, 
manganese, magnesium, chromium, copper, zinc, nickel, iron, aluminum, 
cobalt, gadolinium or other exchangeable ion. Metal ions useful for 
inclusion in compositions for other therapeutic uses may include the 
divalent and trivalent cations selected from the group of magnesium, 
manganese, chromium, zinc and calcium, iron, copper and aluminum. It will 
be obvious to those skilled in the art that various one of the preceding 
metal ions can be used in combination with basic metal chelators, for 
alternative indications than those specified above, and that metal ions 
other than those listed above may, under certain conditions, be useful in 
the uses and indications listed above. 
The compositions described in this invention give surprising and unexpected 
improvements of performance and use which include: 
(1) retained high association of active plus carrier during in vitro 
dialysis and in vivo targeting; 
(2) selective binding of the active plus carrier to induced endothelia at 
sites of disease; 
(3) following intravenous administration, very rapid (2-7 min) localization 
at the diseased site, due to rapid selective endothelial binding, 
envelopment and extravasation of the carrier plus metal chelator across 
disease-induced endothelia (including histologically non-porous 
endothelia); 
(4) widespread uptake throughout the diseased tissue site; 
(5) sustained retention (multiple hours to days) within the diseased site 
in combination with 
(6) rapid plasma clearance (minutes) of the non-targeted fraction; 
(7) moderately slow, polymeric diffusion rates within the diseased tissue 
matrix, allowing differentiation of functional tissue subregions based on 
differences in perfusion of viable and non-viable subregions; 
(8) capacity to selectively image solid tumors or acute vascular and 
myocardial infarcts at body sites, as well as at brain and central nervous 
system sites, with substantially improved selectivity, sensitivity, 
improved delineation of tumor and infarct boundaries at both very short 
and prolonged post-injection intervals, and improved detection of small 
tumor metastases, including those at liver and lung sites. 
Diagnostic and drug enhancement can be made to occur by a number of 
mechanisms, the principal ones being: 
1. Effective TARGETING to tissue sites of disease; 
2. STABILIZATION during both storage and plasma transit; 
3. Prolonged RETENTION at the site of disease, giving a markedly increased 
area under the curve at the tissue site; 
4. RAPID CLEARANCE of the non-TARGETED fraction, thereby reducing 
background signal in imaging applications and reducing normal organ 
exposure and systemic toxicity in therapeutic applications. 
Five further significant advantages of the present compositions and uses 
are: 
1. Simple formulations of active and carrier; 
2. Stabilization of diagnostic and therapeutic actives on the shelf and 
during plasma transit; 
3. Rapid site localization and sustained site retention; 
4. Rapid clearance of the non-targeted fraction; 
5. Availability of low toxicity carbohydrate carriers from natural sources 
and, where needed, modification or derivatization by straightforward 
synthetic means. 
Acidic or anionic saccharides and glycosaminoglycans have unique mechanisms 
of site localization and retention in vivo. They bind to the body's 
endothelial determinants which are selectively induced on the 
microvascular barrier by underlying tissue disease. Previous approaches to 
site targeting were directed at antigenic determinants. However, because 
these determinants are typically located in sequestered sites within the 
tissues, in other words at sites across the endothelial barrier and not 
within the bloodstream and not on its endothelial surface, carriers and 
agents injected into the bloodstream had no effective means to recognize 
and localize in the region of these target antigens. Stated another way, 
previous approaches ignored the major problem of inappropriate carrier 
distribution which resulted from its failure to recognize the vascular 
access codes required for efficient extravasation at disease sites. Hence, 
these carriers failed to effectively load the relevant tissue sites with 
effective concentrations of their bound actives. 
The biological address of a disease site can be viewed in a fashion similar 
to that of a postal address, wherein effective carrier substances must (1) 
first recognize the "state" address of the signal endothelium induced by 
proximal tissue disease; (2) next extravasate and load the "city" address 
of the extracellular tissue matrix with locally effective doses of the 
diagnostic and therapeutic actives; and (3) finally bind and load the 
"street" address of the target cells and antigens. Previous approaches to 
site delivery have attempted to recognize the "street" address without 
first recognizing the "state" and "city" addresses. 
The reason that acidic saccharide and glycosaminoglycan systems work 
substantially better than previous antigen-recognition approaches, is that 
they recognize the newly induced signals which the body uses to attract 
and target white blood cells into sites of tissue disease. When disease 
strikes at a local site, it initiates a cascade of local mediators within 
the tissue matrix and at the endothelial-blood interface which signal the 
blood cells and central body systems that inflammatory and immune cells 
are required within the tissue site. These mediators include cytokines, 
chemoattractants, cytotaxins, induced cell-surface adhesions, selectins 
and integrins, and various tissue-derived and blood-borne, soluble and 
cell-surface procoagulants. White cell accumulation begins within minutes 
and continues over days to weeks, depending on the nature, severity and 
persistence of local disease and the continued generation of tissue 
mediators and trans-endothelial signals. 
As has now been reported and reviewed in detail Ranney (1990); Ranney 
(1992); Bevilaqua et al. (1993); Bevilacqua et al. (1993); Travis (1993); 
Sharon et al. (1993), all incorporated herein by reference!, tumors, 
infarcts, infections, inflammatory diseases, vascular disorders, and other 
focal diseases, characteristically induce the release of such host 
mediators, or cytokines, from resident macrophages and local tissue 
matrix. In certain diseases, alien mediators such as bacterial 
lipopolysaccharides (LPS), viral RNA, and tumor-derived inducers, 
including EMAP II, and chemoattractants may also be released. Although 
additional mediators remain to be elucidated, the principal ones have now 
been defined and include: interleukin 1 (IL-1), tumor necrosis factor 
(TNF), transforming growth factor beta (TGF-beta), Lipopolysaccharide 
(LPS), single and double stranded nucleotides, various interferons, 
monocyte chemoattractant protein (MCP), interleukin 8 (IL-8), interleukin 
3 (IL-3), interleukin 6 (IL-6), tumor-derived inducers and chemoattractant 
peptides (as above), various prostaglandins and thromboxanes. Certain ones 
of the preceding mediators induce the local generation and release of 
metalloproteinases, and these in turn, expose latent tissue binding sites, 
including intact and partially cleaved integrins, RDGS peptides, laminin, 
collagen, fibronectin, and cell-surface core-protein components of 
glycosaminoglycans. 
Cytokines, monocyte chemoattractant protein (MCP), tissue 
metalloproteinases, and proteolytic tissue matrix fragments directly 
induce the local endothelium to become adhesive for circulating white 
blood cells, including neutrophils, monocytes and lymphocytes. The induced 
endothelial adhesive molecules (adhesins) include: P-selectin (gmp-140), 
E-selectin (ELAM-1), intercellular cell adhesion molecule (ICAM-1), 
vascular cell adhesion molecule (VCAM-1), inducible cell adhesion 
molecule, (INCAM-110), von Willebrand's factor (vWF, Factor VIII antigen) 
(see below for disease states which activate these respective types of 
endothelial adhesins). Additional cascades become activated which 
indirectly amplify endothelial adhesiveness. These include (1) coagulation 
factors, especially fibronectin, tissue factor, thrombin, fibrinogen, 
fibrin, and their split products, especially fibronectin split products 
and fibrinopeptide A; (2) platelet-derived factors: platelet activating 
factor (PAF), glycoprotein IIb/IIIa complex; (3) white-cell (a) 
L-selecting, and (b) integrins, including VLA-4 (very late antigen 4); and 
(4) numerous complement factors. 
The preceding pathologic processes and signals are involved, directly or 
indirectly as follows, in the binding and site localization of acidic 
carriers, including acidic saccharides (AC) and glycosaminoglycans (GAGs) 
(Note that in the following outline, potential tissue binding sites are 
designated as "GAGs" and "ACs"). 
1. Local tissue diseases induce local cytokines and mediators, as described 
above. 
2. These cytokines and mediators induce tissue chemoattractants, including 
MCP and IL-8, which comprise a family of arginine-rich, 8 Kd, 
heparin-binding proteins reported to bind GAGs/ACs Huber et al. (1991), 
incorporated by reference herein!; 
3. The cytokines and mediators of No. 1, above, induce the local 
endothelium to express P-selectin, the vascular cell adhesion molecular 
(VCAM-1), inducible cell adhesion molecule (INCAM-110), and von 
Willebrand's factor (vWF, Factor VIII antigen), which are reported binding 
determinants for GAGs/ACs Bevilaqua et al. (1993); Bevilacqua et al. 
(1993)!; P-selectin is reported to bind GAGs Bevilacqua et al. (1993)!; 
4. Integrins, including but not limited to VLA-4, are induced on 
circulating white blood cells, including lymphocytes, during various 
disease processes (see below); VLA-4 has a distinct binding site on the 
(induced) endothelial selectin, VCAM-1 (see No. 3, above); fibronectin, 
which is abundantly present in plasma and also available from tissue 
sites, has a distinct and separate binding site on VLA-4 Elices et al. 
(1990)!; since fibronectin has specific binding sites for GAGs/ACs 
Bevilaqua et al. (1993)!, these amplification steps provide a strong 
additional mechanism for site localization of GAGs/ACs; 
5. The chemoattractants, MCP and IL-8, lymphocyte integrin, VLA-4, platelet 
factor, PAF, and coagulation factors, thrombin, fibronectin and others, 
diffuse from local tissue and blood, respectively, bind to the induced 
endothelial selectins, and amplify adhesiveness and activation at the 
initial endothelial P-selectin sites for GAGs/ACs Elices et al. (1990); 
Lorant et al. (1993) !; 
6. Tissue metalloproteinases become activated and expose new binding sites 
for GAGs/ACs in the tissues which underlie the activated endothelia. These 
new tissue binding sites include as follows Ranney (1990); Ranney (1992); 
Travis (1993); Bevilaqua et al. (1993)!: 
a. fibronectin fragments; 
b. collagen fragments; 
c. laminin fragments; 
d. RGDS peptides; 
e. Exposed core proteins of GAGs; 
7. White blood cells are attracted to the site, become activated and 
release additional proteolytic enzymes, thereby amplifying No. 6 and 
increasing the exposure of binding sites for GAGs/ACs in the tissue 
matrix. 
8. GAG/AC carriers selectively bind the induced and exposed determinants 
listed in Nos. 1-7, above, giving immune-type localization which is 
specific for induced binding sites (lectins) at the lectin-carbohydrate 
level characteristic of white-cell adhesion; 
9. In cases where the carrier substance has multivalent binding to the 
target binding substance, including for example, cases in which the 
carrier is an acidic oligosaccharide or polysaccharide or an acidic 
glycosaminoglycan, multivalent binding of the endothelial surface induces 
rapid extravasation of the carrier and bound active, and results in 
substantially increased loading of the underlying tissue matrix, relative 
to that achieved by antibodies, liposomes, and monovalent binding 
substances, such as hormones and monovalent-binding sugars; 
10. Adhesion of GAGs/ACs to induced and exposed tissue binding sites, 
reduces plasma backdiffusion of GAGs/ACs and their bound actives, thereby 
giving sustained retention within the tissue site; 
11. Controlled release of the diagnostic or drug active from carriers 
comprising GAGs/ACs occurs gradually within the diseased site, thereby 
resulting in targeted controlled release; 
12. Tumor cells, microbial targets and damaged cellular targets within the 
tissue site, may selectively take up the GAG/AC plus bound diagnostic or 
drug active, based respectively, on: induced tumor anion transport 
pathways, microbial binding sites for GAGs/ACs, and proteolytically 
exposed cell-surface core proteins Ranney 07/880,660, 07/803,595 and 
07/642,033!- - - Fe uptake by hepatomas, Cr4S uptake by - - - ); Kjellen 
et al. (1977)! 
13. In cases where the carriers are hydrophilic or essentially completely 
hydrophilic, these carriers cause their bound actives to migrate out 
(permeate) into a small rim of normal tissue around each focus of disease, 
typically comprising a rim about 30-75 um thick; however, such carriers 
undergo selective uptake by abnormal cells within tissue site and 
preferentially avoid uptake by normal cells within the site, thereby 
giving: 
a. In cases of diagnostic imaging applications: very sharp definition of 
the boundary between tumors or infarcts and the surrounding normal 
tissues; 
b. In cases of therapeutic applications: 
(1) protection against spread of disease at the rim; 
(2) relative protection of normal cells within and adjacent to the site of 
disease, from uptake of cytotoxic drugs. 
14. In the case of hydrophilic carriers, including but not limited to 
GAGs/ACs, the non-targeted fraction of active is cleared rapidly and 
non-toxically, thereby minimizing: 
a. in imaging uses, background signal intensity; 
b. in all uses: 
(1) normal organ exposure; and 
(2) systemic side effects. 
Regarding the above outline, the following A. cytokines and mediators; and 
B. selectins, integrins and adhesins are reported to be induced by various 
disease states in addition to that reported for tumors, above Bevilaqua 
et al. (1993)!. Representative non-oncologic induction also occurs as 
follows. 
A. Cytokines and mediators. 
1. MCP: Experimental autoimmune encephalomyelitis in mice Ransohoff et al. 
(1993)!; 
2. IL-8: Neovascularization: Strieter et al. (1992)!; 
3. PAF: Reperfused ischemic heart Montrucchio et al. (1993)!. 
B. Selectins, Integrins and Adhesins. 
1. ELAM-1: 
a. Liver portal tract endothelia in acute and chronic inflammation and 
allograft rejection Steinhoff et al. (1993)!; 
b. Active inflammatory processes, including acute appendicitis Rice et al. 
(1992)!. 
2. VCAM-1: 
a. Simian AIDS encephalitis Sasseville et al. (1992) !. 
b. Liver and pancreas allograft rejection Bacchi et al. (1993)!. 
3. INCAM-110: Chronic inflammatory diseases, including sarcoidosis Rice et 
al. (1991)!. 
4. Integrin, beta 1 subunit cell adhesion receptor: inflammatory joint 
synovium Nikkari et al. (1993)!. 
It is apparent from the above, that broad categories and many specific 
types of focal tissue disease may be addressed by the carriers and actives 
of the present invention, both for diagnostic and therapeutic uses, 
including tumors, cardiovascular disease, inflammatory disease, bacterial 
and viral (AIDS) infections, central nervous system degenerative 
disorders, and allograft rejection. It will also be obvious to those 
skilled in the art, that numerous additional disease states may be 
selectively addressed by the carriers disclosed in this invention. 
The site selectivity of glycosaminoglycans (GAGs) appears to mimic an 
immune mechanism at the level of white-cell targeting rather than antibody 
targeting. Because antibodies have extremely high specificities, they 
characteristically miss major subregions of disease foci (typically as 
great as 60% of tumor cells are nonbinding). Recently, one of the 
GAG-binding determinants of endothelial P-selectin has been identified as 
sialyl Lewis x. Others are in the process of identification. Notably, the 
available nonvalent oligosaccharides specific for sialyl Lewis x suffer 
from two critical problems: 
1. They are exceedingly expensive materials, available only by synthetic or 
semi-synthetic means, and hence, are not cost effective; 
2. They do not bind effectively at diseased sites under in vivo conditions, 
apparently due to the inability as monomeric binding substances to 
displace endogenous interfering substances which are pre-bound at these 
sites. 
There are two apparent benefits of the relatively broader range of GAG 
specificities and redundancy of GAG binding sites present on diseased 
endothelium and tissue matrix: 
1. GAGs allow a broader range of tumors and diseases to be targeted than 
that possible with antibodies (which typically target only a subset of 
histologic types -- -- -- even within a given class of tumor, and hence, 
are typically ineffective from both a medical and cost/development 
standpoint); 
2. GAGs are projected to be effective over a greater time interval, from 
early onset of disease to progression and regression. 
Despite the broader targeting specificity of GAGs over antibodies, their 
favorable clearance and avoidance of uptake by normal cells reduce 
systemic and local toxicities, even though more than one type of disease 
site may undergo targeted accumulation of the diagnostic/drug within its 
extracellular matrix. 
The polymeric and multivalent binding properties of GAGs both are very 
important for optimal site localization of the attached diagnostic/drug. 
GAG molecular weights of ca. 8,000 to 45,000 MW are important in order to: 
1. Restrict initial biodistribution of the diagnostic/drug to the plasma 
compartment and thereby maximize the quantity of agent available for site 
targeting; 
2. Displace endogenous interfering substances which are pre-bound to 
diseased endothelium; 
3. Induce active endothelial translocation of the GAG-diagnostic/drug into 
the underlying tissue matrix; 
4. Afford rapid clearance and markedly reduced side effects of the attached 
actives. 
SUMMARY OF THE INVENTION 
The present invention encompasses novel agents comprising cationic or 
chemically basic metal chelators in association with hydrophilic carriers 
of anionic or chemically acidic saccharides, sulfatoids and 
glycosaminoglycans. In certain embodiments of the invention, the agents 
also comprise chelated metals and metal ions. The binding of the metal 
chelators to the carriers is stabilized by covalent or non-covalent 
chemical and physical means. In some embodiments, novel non-covalently 
bound compositions give uniquely high payloads and ratio of metal chelator 
to carrier, ranging from a low of about 15% metal chelator by weight, to a 
characteristic range of 70% to 90% metal chelator by weight. Specific 
embodiments comprise deferoxamine, ferrioxamine, iron-basic porphine, 
iron-triethylenetetraamine, gadolinium DTPA-lysine, gadolinium DOTA-lysine 
and gadolinium with basic derivatives of porphyrins, porphines, expanded 
porphyrins, Texaphyrins and sapphyrins as the basic or cationic metal 
chelators, which are in turn, bound to acidic or anionic carriers, 
including one or more of acidic or anionic saccharides, and including 
sulfated sucrose, pentosan polysulfate, dermatan sulfate, oversulfated 
dermatan sulfate, chondroitin sulfate, oversulfated chondroitin sulfate, 
heparan sulfate, beef heparin, porcine heparin, non-anticoagulant 
heparins, and other native and modified acidic saccharides and 
glycosaminoglycans. 
Methods of magnetic resonance image (MRI) contrast enhancement are a 
particular embodiment of the present invention which confirm very rapid, 
carrier-mediated, site-selective in vivo localization and sustained site 
retention of metal-chelator compositions, based on stable binding of the 
metal chelator and carrier during in vivo plasma transit, allowing site 
localization following intravenous administration. Rapid and selective 
endothelial-site binding, facilitated rapid extravasation into underlying 
tissue sites, site accumulation, sustained site retention, together with 
rapid clearance of the non-site-localized fraction are also demonstrated 
by the use of the compositions of the present invention in the selective 
MRI contrast enhancement of tumors and cardiovascular infarcts. 
Surprising and unexpected improvements of selectivity, mechanism of 
localization and cellular uptake, and MRI contrast sensitivity are shown 
for metal chelates having standard paramagnetic potencies. Further 
advantages of the use of the compositions and methods of the present 
invention are delineated in the examples (infra) including special 
histologic staining evidence which confirms the site-selective endothelial 
binding, extravasation, tissue matrix accumulation and cellular uptake 
mechanism. Selective localization and MRI imaging efficacy are also shown 
to occur when paramagnetic metal chelator actives are administered in 
carrier-bound form but not in free form. 
In its most general embodiment, the present invention is an agent 
comprising a chelator for metal ions, said chelator having a cationic 
group and being bound to an anionic, hydrophilic carrier. In alternate 
embodiments, the chelator for metal ions which has a cationic group is 
bound to an anionic, hydrophilic carrier by non-covalent electrostatic 
binding. And, in certain alternate embodiments the invention comprises an 
agent comprising a basic chelator for metal ions, said chelator having a 
cationic group and being covalently bound to an anionic, hydrophilic 
carrier. In the particular embodiments of the invention in which the 
chelator is not covalently bound to the carrier, the said chelator may be 
basic. 
In certain embodiments of the present invention, the agent which comprises 
a chelator for metal ions and having a cationic group bound to an anionic 
hydrophilic carrier may further comprise a chelated metal ion, and in 
particular it may further comprise a paramagnetic metal ion. The agents of 
the present invention, in particular those which comprise the chelator for 
metal ions non-covalently bound to the carrier may be further defined as 
being at least about 15 weight percent chelator. Preferably, the chelator 
has a formation constant for paramagnetic metal ions of at least about 
10.sup.14. 
Those agents of the present invention which comprise a metal ion will 
preferably comprise a metal ion selected from the group consisting of 
iron, manganese, chromium, copper, nickel, gadolinium, erbium, europium, 
dysprosium and holmium. In certain embodiments, the agents of the present 
invention may even comprise a metal ion selected from the group consisting 
of boron, magnesium, aluminum, gallium, germanium, zinc, cobalt, calcium, 
rubidium, yttrium, technetium, ruthenium, rhenium, indium, iridium, 
platinum, thallium and samarium. It is understood that other metal ions 
which are functionally equivalent to the listed metal ions are also 
included and would fall within the scope and spirit of the presently 
claimed invention. 
In certain preferred embodiments of the invention, the agents comprise a 
carrier wherein said carrier is an acidic saccharide, oligosaccharide, 
polysaccharide or glycosaminoglycan. The carrier may also be an acidic 
glycosaminoglycan or sulfatoid. In particular, the carrier may be, but is 
not limited to heparin, desulfated heparin, glycine-conjugated heparin, 
heparan sulfate, dermatan sulfate, chondroitin sulfate, hyaluronic acid, 
pentosan polysulfate, dextran sulfate, sulfated cyclodextrin or sulfated 
sucrose. 
In certain embodiments of the invention, the chelator is a chelator of iron 
ions. Preferably the chelator is a hydroxamate, and more preferably it is 
deferoxamine. In certain preferred embodiments the chelator together with 
the metal ion is ferrichrome, ferrioxamine, enterobactin, ferrimycobactin 
or ferrichrysin. In a particularly preferred embodiment, the chelator is 
deferoxamine, the carrier is heparin, or a heparin fragment and the agent 
further comprises iron(III). In an alternate embodiment, the chelator is 
deferoxamine and the carrier is dermatan sulfate or a dermatan sulfate 
fragment and the agent may further comprise chelated iron(III). 
In a certain embodiment, the invention may also comprise deferoxamine bound 
to a carrier selected from the group consisting of heparin, heparan 
sulfate, dermatan sulfate or chondroitin sulfate, and may further comprise 
a metal ion. The agents of the present invention may also comprise a 
chelator which is a porphine, porphyrin, sapphyrin or texaphyrin and which 
may further comprise a metal ion, and preferably an iron ion or a 
gadolinium ion. 
In a particularly preferred embodiment the agent of the present invention 
may comprise a chelator which is 
5,10,15,20-Tetrakis(1-methyl-4-pyridyl)-21H,23-porphine, a carrier which 
is heparin and a chelated iron ion. In certain embodiments, the chelator 
may also be a polyaminocarboxylate or macrocyclic, and preferably a basic 
or amine derivative of diethylenetriaminetetraacetate, or more preferably 
a basic or amine derivative of 
1,4,7,10-tetraazacyclododecane-N,N',N","'-tetraacetate (DOTA). In the 
agents of the present invention, the carrier may also be defined further 
as being complementary to endothelial determinants selectively induced at 
disease sites. 
In a certain embodiment, the present invention is an image-enhancing agent 
or spectral-enhancing agent to enhance images arising from induced 
magnetic resonance signals, the agent comprising ferrioxamine covalently 
conjugated to heparin by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, 
N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline, or carbonyldiimidazole. 
Alternatively, the invention is a spectral-enhancing agent to enhance 
images arising from induced magnetic resonance signals, the agent 
comprising Gd(III)diethylenetriaminepentaacetate covalently conjugated to 
one of heparin, dermatan sulfate or chondroitin sulfate. In another 
alternative, the invention is an agent for in vivo imaging, the agent 
comprising a basic chelator for metal ions and chelated metal ion, said 
chelator being bound by non-covalent electrostatic binding to a 
hydrophilic carrier selected from the group consisting of heparin, 
desulfated heparin, glycine-conjugated heparin, heparan sulfate, dermatan 
sulfate, chondroitin sulfate, hyaluronic acid, pentosan polysulfate, 
dextran sulfate, sulfated cyclodextrin or sulfated sucrose. The agent for 
enhancing body imaging preferably comprises deferoxamine, chelated Fe(III) 
and a glycosaminoglycan carrier bound to said deferoxamine and more 
preferably the glycosaminoglycan carrier is dermatan sulfate, and/or the 
Fe(III) is a radiopharmaceutical metal ion, and most preferably the 
radiopharmaceutical metal ion is .sup.59 iron or .sup.67 gallium. 
In an alternate embodiment, the invention is an agent for enhancing body 
imaging, the agent comprising diethylenetriaminepentaacetate-lysine, 
chelated Gd(III) and a glycosaminoglycan carrier bound to said 
diethylenetriaminepentaacetate-lysine. Alternatively, the invention is an 
agent for enhancing body imaging, the agent comprising DOTA-lysine, 
chelated Gd(III) and a glycosaminoglycan carrier bound to said 
1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetate-lysine 
(DOTA-lysine). In a particular embodiment, the invention is an agent 
comprising ferrioxamine bound by non-covalent electrostatic binding to 
dermatan sulfate. 
It is understood that any of the agents of the present invention as 
described in the above paragraphs or in the appended claims may be defined 
further as being in a combination with at least one of a buffer, 
saccharide, sulfated saccharide, or salt, to produce an osmotic strength 
suitable for parenteral administration, and as being an aqueous solution 
or a lyophilized or dry preparation suitable for aqueous reconstitution 
having the desired osmotic strength, and wherein said agent is aseptic or 
sterile. 
Another embodiment of the invention is a method of enhancing magnetic 
resonance images or spectra in vertebrate animals comprising administering 
to said animal an effective amount of an agent of the invention which 
comprises the metal ion chelator, the carrier as described and a 
paramagnetic ion. In particular, the invention is a method of enhancing in 
vivo images arising from induced magnetic resonance signals, comprising 
the steps of administering to a subject an effective amount of an agent of 
the present invention which comprises a paramagnetic ion, exposing the 
subject to a magnetic field and radiofrequency pulse and acquiring an 
induced magnetic resonance signal to obtain a contrast effect. 
In an alternative embodiment, the invention is a method of enhancing in 
vivo images, comprising the steps of administering to a subject an 
effective amount of an agent of the present invention which comprises a 
chelated metal ion, exposing the body to ultrasound or X-rays and 
measuring signal modulation to obtain a contrast effect. 
In another embodiment, the invention is a method of obtaining in vivo body 
images comprising administering to a subject an effective amount of an 
agent of the invention which comprises a metal ion wherein the metal ion 
is a radioisotope and measuring scintigraphic signals to obtain an image. 
In another embodiment, the invention is a method of treating vascular 
disease, comprising administering to a subject a therapeutically effective 
amount of an agent of the present invention, and preferably an agent which 
comprises a metal ion.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
The many innovative teachings of the present invention will be described 
with particular reference to the presently preferred embodiments, wherein 
these innovative teachings are advantageously applied to the particular 
issues of in vivo T1-Type MRI image contrast enhancement by site-selective 
localization and sustained site retention of paramagnetic metal chelates 
according to optimal spatial and kinetic profiles at the site, while 
simultaneously enhancing clearance and minimizing toxicity of the 
non-localized dose fraction. However, it should be understood that this 
principal embodiment is only one example of the many advantageous uses of 
the innovative teachings herein. For example, the various types of 
innovative compositions and methods disclosed herein can alternatively be 
used to selectively localize and enhance clearance of radionuclide imaging 
agents, X-ray contrast agents, ultrasound-acoustic image enhancing agents 
and a wide spectrum of therapeutic agents which are based on site delivery 
of metal chelates and in situ chelation of endogenous body metals. Of 
special interest to the therapeutic agents and uses embodied herein, are 
actives and indications useful in oncotherapy, cardiovascular infarcts, 
restenosis, atherosclerosis, acute thrombosis, microvascular disease, 
inflammation, and any other tissue diseases which have as part of their 
development or progression, a vascular component amenable to modulation by 
the novel teachings, compositions and uses described herein. Hence, it 
will be obvious to those skilled in the art, that numerous additional 
compositions and uses are uniquely enabled by the present invention. The 
following examples are presented to illustrate preferred embodiments of 
the present invention, their uses in MRI contrast enhancement. These 
examples are purely illustrative, and do not in any way delimit the full 
scope of the present invention. 
The present invention specifically describes the preparation and 
utilization of novel contrast agents for magnetic resonance imaging. These 
novel contrast agents consist of paramagnetic metal chelates, as 
distinguished from metal-atom complexes, wherein the presently disclosed 
chelates are bound to glycosaminoglycans (GAG). Binding of the metal 
complex to the GAG may take the form of, but is not limited to, 
electrostatic interactions (ion-paired), hydrogen-bonding, Van der Waals 
interactions, covalent linkages, or any combination of these interactions. 
Following parenteral administration of these metal 
complex-glycosaminoglycan formulations, the technology described herein 
utilizes a biocompatible carrier molecule to deliver an associated 
biologically active substance to sites of vascular injury. 
The present invention provides substantially improved MRI image and 
spectral enhancement compositions and methods, whereby the capacity of MRI 
hardware systems to detect tumors, cardiovascular diseases, and other 
diseases with a vascular or endothelial adhesive component are greatly 
enhanced. These improvements are presently accomplished by introducing a 
chelated paramagnetic metal ion selectively into tissue sites of interest, 
inducing selective (local) modulation of T1-Type, paramagnetic relaxation 
of water protons or other diffusible nuclei present within the site which 
are susceptible to orientation by fixed and gradient magnetic fields and 
to pulsed re-orientation by radiofrequency fields of appropriate resonant 
frequencies, thereby giving rise to detectable modulations of induced 
magnetic resonance signals, in the forms of either image contrast 
enhancement or spectral enhancement. 
Past disclosures (Ranney: U.S. Ser. No. 07/880,660, filed May 8, 1992, U.S. 
Ser. No. 07/863,595 filed Apr. 3, 1992, now U.S. Pat. No. 5,214,661 and 
U.S. Ser. No.07/642,033 filed Jan. 1, 1991! have dealt with the binding of 
magnetic agents which required: (a) magnetic potencies greater than that 
of the most potent single metal ion, gadolinium(III); (b) intramolecularly 
coupled, polyatomic metal-atom complexes stabilized by non-bridged ligands 
which have a stronger potential for chemical and physical instability than 
the stably, bridged-ligand chelated metal ions disclosed herein; and (c) 
divalent cationic charge on the "superparamagnetic" active for binding to 
anionic carriers, versus the presently disclosed requirement for only a 
monovalent cationic charge on paramagnetic metal chelator actives. It is 
understood, that for MRI uses, the metal chelator will also comprise an 
appropriate paramagnetic metal ion, for example, preferably iron(III) or 
gadolinium (III), however, for certain other diagnostic and therapeutic 
compositions and uses, the presently disclosed metal chelators may either 
comprise or avoid an appropriate metal ion. For the presently preferred 
MRI applications, basic metal chelators and metal chelators with 
electrophilic properties at formulation pH's are preferred, for example, 
ferrioxamine Crumbliss, 1991!, basic or amine derivatives of the 
polyaminocarboxylate chelator, diethylenetriaminepentaacetate (DTPA), and 
basic or amine derivatives of the macrocyclic chelator, 
1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetate (DOTA) Li et al. 
1993; Brechbiel et al. 1986!. In certain instances and with certain potent 
carriers bound to these and related actives, site localization may be so 
pronounced that the inherent potency (in vitro paramagnetic R1) of the 
paramagnetic metal ion may not be crucial to obtaining optimal 
site-localized image contrast or spectral enhancement effects. Hence, the 
present invention discloses pronounced T1 image contrast effects for the 
basic metal chelate, ferrioxamine, which by virtue of chelated Fe(III) 
ions, has a potency, or R1 relaxivity, of about 1.6-1.8 mmol.sec!-1. 
Alternatively, basic metal chelates of Gd(III) maybe expected under 
certain but not all in vivo conditions, to have a potentially greater 
relaxivity, due to its greater in vitro R1 of about 4.0-4.3 mmol.sec!-1 
when chelated by DTPA, and potentially moderately higher when chelated by 
DOTA Geraldes et al. 1985!. Alternative metal ions may preferably include 
the divalent or trivalent cations, manganese, chromium and dysprosium; and 
less preferably, those ions of copper, nickel, erbium, europium, and 
holmium. 
Preferred chelators of the present invention include those with a formation 
constant of at least about 10.sup.14 for strongly paramagnetic metal ions 
disclosed above, and including a basic or cationic group. These chelators 
preferably include ferrioxamine, basic or amine derivatives of DOTA, DTPA, 
porphines, porphyrins, sapphyrins or texaphyrins, which can preferably 
chelate Fe(III) and most preferably chelate Gd(III), as well as bind by 
principally paired-ion (electrostatic) means to the acidic groups of 
acidic carriers. For example, certain texaphyrins have an expanded 
macrocyclic ring which may, in certain instances, stably chelate Gd(III) 
Sessler et al. '065; Sessler et al. '720; Sessler et al. '498, 
incorporated by reference herein!. Whereas texaphyrins and sapphyrins are 
not exemplified in the present invention, it will be obvious to those 
skilled in the art, from the references cited just above, and from the 
presently disclosed and exemplified Fe(III) chelator, 
5,10,15,20-Tetrakis(1-methyl-4-pyridyl)-21-23-porphine, that the related 
texaphyrins and sapphyrins and their basic, cationic and amine 
derivatives, as well as the presently disclosed porphine derivative and 
its analogues and basic, cationic and amine derivatives, would be included 
under the disclosures and teachings of the present invention, and may be 
used in combination with the presently disclosed acidic carriers. There 
are hybrid considerations of, among others: (a) paramagnetic potency of 
the metal chelate; (b) binding stability to the acidic carrier; and (c) 
formulation compatibility; and (d) biocompatibility and clearance in vivo. 
Hydrophilic chelators and carriers are usually, but not always preferred, 
due to their typically favorable formulation properties (absence of 
aggregation), biodistribution properties (absence of generalized binding 
to hydrophobic plasma and cell-membrane constituents during the process of 
localization); and clearance plus toxicity advantages. Alternative 
chelators may include the hydroxamates, ferrichrome, enterobactin, 
ferrimycobactin, ferrichrysin, and their basic or amine derivatives, all 
derivatives being defined as subsumed under the parent chelators listed 
above. 
Preferred carriers include monomeric, oligomeric and polymeric substances 
which contain or comprise anionic or acidic groups defined at the pH's 
used for formulation. These typically contain or comprise groups of 
carboxylate, and more preferably, the even more strongly acidic groups of 
phosphate, and most preferably, sulfate. Preferred carriers include, but 
are not limited to an acidic saccharide, oligosaccharide, polysaccharide, 
glycosaminoglycan or sulfatoid, typically of bacterial or semi-synthetic 
origin, or derivatives or modifications or fragments of the preceding 
substances, all defined herein as being subsumed under the names of the 
parent substances and categories. Hence, preferred carriers include the 
following: heparin, desulfated heparin, glycine-conjugated heparin, 
heparin sulfate, dermatan sulfate, chondroitin sulfate, pentosan 
polysulfate, and sulfated sucrose, including sucrose octasulfate, and any 
derivative, modification or modified form thereof. Less preferably for 
typical MRI formulations and uses, are include the carriers of sulfated 
cyclodextrin, dextran sulfate and hyaluronic acid, although any of these 
may be particularly suitable for certain specific diagnostic or 
therapeutic formulations and uses. 
In all cases reported and tested, non-covalent binding of the basic amine 
chelator to the acidic carrier gives payloads of active agent which are 
markedly higher than those afforded by covalent conjugation. For example, 
and preferably, ferrioxamine and Gd(III) DTPA-lysine are bound to their 
acidic glycosaminoglycan carriers at weight ratios of .gtoreq.70%. 
Alternative covalent active-carrier conjugates may be preferred in certain 
instances, and preferred examples thereof are shown for MRI applications. 
Specific embodiments of the present invention which have been tested in 
vivo, include, but are not limited to the presently exemplified preferred 
embodiments of: (a) deferoxamine, (b) ferrioxamine and (c) 
Gd(III):DTPA-lysine basic metal chelates bound by most preferably 
non-covalent means, and also preferably by covalent means, as exemplified 
below, to acidic glycosaminoglycans, including preferably, dermatan 
sulfate, chondroitin sulfate, heparan sulfate, and heparin, which include 
by definition, any derivative or modification thereof, including 
oversulfation and modification undertaken to reduce anticoagulant 
activities or provide improved site binding, enhanced clearance or other 
desired formulation or in vivo properties. Alternative preferred Agents 
obvious from the present disclosures, to those skilled in the art, may 
induce arginine and histidine basic derivatives of DTPA and DOTA, and also 
of the various texaphyrins, sapphyrins, porphines, porphyrins, EHPG, and 
by definition, most preferably for MRI applications, comprising their 
Gd(III) and Fe(III) metal-ions, an also preferably comprising their 
alternative paramagnetic metal ion chelates, as disclosed above. 
The present invention describes the preparation and utilization of a novel 
MRI contrast agent for enhancement of solid tumors and cardiovascular 
infarcts. The contrast agents consist of cationic or basic paramagnetic 
metal complexes in association with strongly acidic, including 
polysulfated carriers, and including preferably glycosaminoglycans. It 
would be obvious to those skilled in the art that any acidic 
glycosaminoglycan can be used. Of the paired-ion systems described below, 
most notably are those consisting of ferrioxamine with glycosaminoglycans 
and DTPA-lysine with glycosaminoglycans. 
It is envisioned that alternative diagnostic and therapeutic compositions 
and applications may be carried out using compositions substantially 
similar to those disclosed above. For example, alternative metal ions may 
be chelated for purposes of metal-ion exchange at the site. Hence, the 
present formulations may contain or comprise metal ions of manganese, 
aluminum, germanium, zinc, cobalt, calcium, platinum, or others. 
Alternatively, for purposes of radiation and radionuclide therapy, such 
compositions may contain or comprise metal ions of boron, cobalt, 
rubidium, yttrium, technetium, ruthenium, rhenium, indium, iridium, 
thallium, samarium or others. Specifically, and in some cases preferably, 
.sup.59 Fe and .sup.67 Ga Hashimoto et al. 1983; Janoki et al. 1983! may 
be substituted as radionuclide forms of the non-radioactive metal ions, 
for purposes of nuclear medical imaging of tumors, thrombi, and other 
biomedical imaging purposes. 
The preceding discussion is presented to specify major aspects of the 
invention and their use in in vivo diagnostic and therapeutic 
applications, however, to those skilled in the art many additional and 
related compositions and methods of use will be obvious from this 
preceding discussion and are encompassed by the present invention. 
TABLE 1 
______________________________________ 
Advantages of Metal Ion Chelator and 
Anionic, Hydrophilic Carrier 
Selective 
Technology 
MRI Agent Antibodies 
PEG Liposomes 
______________________________________ 
Property 
Drug Payload 
High 77.5% 
Very Low Low 10-30% 
Low 
5% 15-20% 
Localization 
Yes Very Low No No 
In Tissue 
Sites 
Selectivity 
Broad Narrow None None 
Immune Immune 
(CHO- (Ab- 
lectin) antigen) 
Time to Very Rapid 
Slow Slow Very Slow 
Target (several (several (many hrs) 
(hrs-days) 
mins) hrs) 
Time to Rapid Very Slow 
Very Slow 
Extremely 
Clear Plasma Slow (RES) 
& Body 
Applications 
Broad Narrow Narrow Narrow 
(Tissue (Intravasc 
(Enzymes) 
(RES) 
Sites) ular) 
______________________________________ 
The following examples are included to demonstrate preferred embodiments of 
the invention. It should be appreciated by those of skill in the art that 
the techniques disclosed in the examples which follow represent techniques 
discovered by the inventor to function well in the practice of the 
invention, and thus can be considered to constitute preferred modes for 
its practice. However, those of skill in the art should, in light of the 
present disclosure, appreciate that many changes can be made in the 
specific embodiments which are disclosed and still obtain a like or 
similar result without departing from the spirit and scope of the 
invention. 
EXAMPLE 1 
Preparation of Deferoxamine Free Base and Use in Formulation of 
Ferrioxamine 
The free base of deferoxamine is used in certain instances, in order to 
minimize the residual salt content present in final formulations which 
utilize deferoxamine as a basic metal chelator. In these instances, 
deferoxamine is precipitated out of aqueous salt solutions by the addition 
of 2 N KHCO.sub.3, as previously reported Ramirez et al. (1973), 
incorporated by reference herein!. A saturated solution of deferoxamine 
(320 mg/mL at 25.degree. C.) is prepared by dissolving 4.0 g of 
deferoxamine mesylate salt in 12.5 mL of pharmaceutical-grade water. The 
solution is cooled to 4.degree. C. in an ice bath and 2.5 mL of 2.0N 
KHCO.sub.3 added. The glass container is scratched with a stainless steel 
spatula to initiate precipitation. The precipitate is collected by 
centrifugation, washed repeatedly with ice cold water, and filtered. The 
crude deferoxamine free base is purified by sequential recrystallization 
from hot methanol. The resulting pure deferoxamine free base is dried 
under a stream of nitrogen. The infrared spectrum of the deferoxamine as 
prepared is consistent with that referenced above. 
Ferrioxamine is formulated from the deferoxamine free base by addition of 
ferric chloride at stoichiometric molar ratios of Fe(III) to deferoxamine 
free base. This results in chelated iron and minimizes residual mesylate 
and chloride ions. 
EXAMPLE 2 
Preparation of Ferrioxamine-Iron (III) Chelate 
Batch quantities of the Fe(III) chelate of deferoxamine are prepared as 
follows. Deferoxamine mesylate (methanesulfonate) (Ciba-Geigy Limited, 
Basel, Switzerland), 390 g, is dissolved in pharmaceutical-grade water. 
Alternatively, the chloride salt of deferoxamine may be used. A highly 
purified slurry of ferric iron in the form of Fe(O)OH (13.44% w/v of 
Fe(O)OH particles, Noah Technologies Corporation, San Antonio, Tex.), 
372.9 g is suspended in 1899 mL of water and added to the deferoxamine 
with constant stirring. The resulting suspension is heated to 60.degree. 
C. for between 1 and 24 hours and the pH adjusted periodically to between 
6.5 and 7.9 by addition of 0.10N NaOH. Formation the ferrioxamine complex 
is evidenced by development of an intense deep reddish-brown color to the 
solution. Stoichiometric chelation of Fe(III) with deferoxamine is 
confirmed by in-process UV-Visible absorbance spectroscopy at 430 nm, 
against stoichiometrically chelated ferrioxamine standards. The batch 
solution is cooled to room temperature and centrifuged at 4500 rpm 
(.apprxeq.2500 g) for 15 minutes to remove any unreacted or aggregated 
Fe(O)OH. This final batch volume is adjusted as desired, typically to a 
final volume of 2600 mL. Any remaining trace amounts of unreacted Fe(O)OH 
are removed and the solution also made aseptic, by passing the supernatant 
through a 0.22 .mu.m Millipore GV-type filter in a Class 100 laminar flow 
hood. The resulting batch is stored at 4.degree. C. in an autoclaved, 
sealed glass container until further use (see Examples below). The final 
concentration of ferrioxamine (DFe) is determined once again by UV-Visible 
absorbance spectrophotometry at 430 nm. 
EXAMPLE 3 
Preparation of the Basic Amine Chelator: 
Diethylenetriaminepentaacetate-Lysine (DTPA-Lys) 
DTPA, 500 mg, is dissolved in 20 mL of pharmaceutical-grade water and 
heated to 60.degree. C. L-Lysine hydrochloride powder, 931 mg, is added 
with constant stirring until dissolved. Alternatively, 
N-epsilon-t-BOC-L-lysine can be used to prevent reaction of the 
carbodiimide intermediate at the lysine epsilon amino group (see below), 
and when used, is dissolved in dimethylformamide:water (50:50, w/v). The 
solution pH is adjusted to 4.75 by addition of 0.1N HCl. The water-soluble 
carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide HCl (EDC), 
732.5 g, is dissolved in 2 mL water and its pH also adjusted as above. 
This EDC solution is added dropwise to the DTPA+lysine solution mixture 
(above) over 1 hour at 22.degree. C. with constant stirring and periodic 
adjustment of pH to 4.75, and the reaction allowed to proceed to 
completion over 2 more hours. When N-epsilon-t-BOC-L-lysine is used (see 
above), the N-epsilon-t-BOC group is hydrolyzed at this step, by 
acidification with hydrochloric acid to a pH of between 1.0 and 2.0, and 
stirring for 30-60 min. The pH is readjusted to 4.75 as needed, and the 
reaction solution is concentrated down to 5 mL by rotary evaporation at 
60.degree. C., and the DTPA-lysine (DTPA-Lys) derivative is precipitated 
by addition of 3 volumes of ethanol. Note: under these conditions, the 
ethanol:water ratio used, maintains the solubility of all individual 
substrates (above). The resulting precipitate is harvested by 
centrifugation at 2,500.times.g, washed with ethanol, re-centrifuged, and 
dried over a stream of dry nitrogen. Covalent conjugation of lysine to 
DTPA is confirmed by infrared (IR) spectroscopy. The resulting reaction 
product has a faint yellow color. 
EXAMPLE 4 
Preparation of the Gadolinium(III) Metal Chelate of DTPA-Lys: 
gadolinium:DTPA-Lys Gd(III):DTPA-Lys! 
The gadolinium(III) chelate of DTPA-Lys, namely Gd(III):DTPA-Lys, is 
prepared by dissolving a known quantity of DTPA-Lys in water and adding a 
stock solution of gadolinium chloride, prepared at 0.1-1.0M, as needed, 
until a stoichiometric quantity of Gd(III) has been added. The pH is 
adjusted to 7.0 by addition of 1.0N NaOH. Alternatively, gadolinium oxide 
can be added and the reaction mixture stirred for 24 hours. In the case of 
gadolinium oxide, neutralization with 1.0N NaOH is not needed. Each batch 
of Lys-DTPA conjugate is pre-titrated and the final chelation product 
checked for stoichiometric addition of Gd(III), using a standard xylenol 
orange titration method Lyle et al. (1963)!, and further confirmed by 
quantitative ICP atomic absorption spectroscopy for gadolinium. The 
resulting Gd(III):DTPA-Lys is precipitated by addition of ethanol (3 
volumes per volume of water), and the precipitate collected by 
centrifugation. This precipitate is rewashed with ethanol and centrifuged 
(as above), washed with acetone plus centrifuged, and the collected 
precipitate dried over a stream of dry nitrogen. The resulting product 
continues to have the same faint yellow color as noted in Example 3. 
EXAMPLE 5 
Preparation of Paired-ion Agents of Ferrioxamine Bound to Dermatan Sulfate 
Carriers; and Ferrioxamine to Depolymerized Dermatan Sulfate Carrier 
Ferrioxamine:dermatan sulfate paired-ion agents are prepared by mixing 
appropriate ratios of the water solutions of ferrioxamine (see Example 2, 
above) with either: (a) dermatan sulfate of modal MW between approximately 
5,000 daltons and 45,000 daltons (Opocrin, S.p.A., Modena, Italy; and 
Scientific Protein Laboratories, Waunake, Wis.); or (b) depolymerized 
dermatan sulfate of modal MW between approximately 2,000 daltons and 5,000 
daltons (Opocrin S.p.A., Modena, Italy). A range of ratios of ferrioxamine 
to dermatan sulfate are prepared between a low of 1:99 (wt %) of 
ferrioxamine:dermatan sulfate or depolymerized dermatan sulfate; and a 
high of 30:70 (wt %) of ferrioxamine: dermatan sulfate or depolymerized 
dermatan sulfate). Using 0.1 to 1.0N NaOH, the pH of the mixture is 
adjusted to between 5.5 and 8, the mixture is stirred continuously for 0.5 
to 72 hours and the pH re-adjusted between 5.5 and 8, and typically to 
7.5. This ferrioxamine:dermatan mixture is passed through a 0.22 .mu.m 
filter to remove any residual insoluble iron oxides and hydroxides, and to 
render the liquid agent aseptic. The aseptic agent is stored either as a 
liquid at 4.degree. C., or as a lyophilized powder (see below). Further 
processing is carried out on the liquid, by filling into glass vials and 
autoclaving at 120.degree. C. for 15 minutes. Alternatively, further 
processing is carried out on the liquid by filling into glass vials, 
freezing at -50.degree. C., and lyophilization to give an aseptic 
lyophilized powder. The lyophilized vials are reconstituted by adding 
sterile water and hand mixing for 1 to 5 minutes, to give a reconstituted 
liquid of desired concentration which is ready for injection. The 
resulting concentrations of ferrioxamine and dermatan sulfate are measured 
and vial quantities confirmed by standard reverse-phase HPLC and 
macromolecular size exclusion HPLC methods, respectively. 
Multiple batches of Ferrioxamine:Dermatan Sulfate Agent have been prepared. 
In vitro test results for a representative batch are as follows: 
ferrioxamine:dermatan sulfate ratio: 77.5:22.5 (w/w); solubility of agent, 
550 mg/mL; water:octanol partition, 17,600 (.+-.2,750):1; concentration of 
ferrioxamine, 0.166 mmol/mL; concentration of dermatan sulfate, 32 mg/mL; 
molecular weight of dermatan sulfate, MN=18,000 daltons; 
sulfate/carboxylate ratio of dermatan sulfate, 1.0.+-.0.15; ferrioxamine 
and dermatan purities, nominal .+-.10%; pH, 6.5-7.9; viscosity, 3.8-4.2 
centipoise; osmolality, 475-525 mOsm/Kg; R1, 1.5-1.8 mmol.sec!-1; 
oversized particles, within USP guidelines for small-volume parenterals; 
Anticoagulant activity, less than 4.5 U.S.P. Units/mg (modified USP XXII 
assay); binding of ferrioxamine active to dermatan carrier, at least 92% 
retained (dialysis for 3 hours against 200 volumes, 500 daltons molecular 
weight cutoff). 
In vitro stability of Ferrioxamine:Dermatan Sulfate Agent under accelerated 
conditions, indicate the following. (a) The liquid form is stable, by the 
preceding physicochemical and HPLC parameters for longer than 6 months at 
4.degree. C., 22.degree. C. and 40.degree. C.; is slightly unstable at 2 
to 6 months at 60.degree. C., and degrades significantly within 1 to 3 
days at 80.degree. C. (b) The liquid form can be autoclaved as above, with 
less than 3% degradation of ferrioxamine. (c) The lyophilized form is 
stable with respect to all parameters (above), including oversized 
particles; and is projected to be stable over storage periods of multiple 
years. 
EXAMPLE 6 
Preparation of Paired-ion Agents of Ferrioxamine Bound to Heparin 
Ferrioxamine:dermatan sulfate paired-ion agents are prepared by mixing 
appropriate ratios of water solutions of ferrioxamine (as in Example 5, 
above) with (a) beef lung heparin of modal MW between approximately 8,000 
daltons; and (b) porcine heparin of modal MW between approximately 10,000 
daltons and 20,000 daltons. A range of ratios of ferrioxamine to heparin 
or heparin fragment are prepared between a low of 1:99 (wt/wt) of 
ferrioxamine:heparin or heparin fragment; and a high of 30:70 (wt of 
ferrioxamine:fragment. Using 0.1 to 1.0N NaOH, the pH of the mixture is 
adjusted to between 5.5 and 8, the mixture is stirred continuously for 0.5 
to 72 hours and the pH re-adjusted between 5.5 and 8. This 
ferrioxamine:heparin mixture is passed through a 0.22 .mu.m filter to 
remove any residual insoluble iron oxides-hydroxides and render the liquid 
agent aseptic. The aseptic agent is stored at 4.degree. C. As indicated, 
further processing is carried out by filling the aseptic liquid in glass 
vials, followed by freezing and lyophilizing, to render the agent as an 
aseptic lyophilized powder. The lyophilized vials are reconstituted by 
adding sterile water and hand mixing for 1 to 5 minutes, to give a 
reconstituted liquid of desired concentration which is ready for 
injection. The resulting concentrations of ferrioxamine and heparin are 
measured and vial quantities confirmed by standard reverse-phase HPLC and 
macromolecular size exclusion HPLC methods, respectively. 
EXAMPLE 7 
Preparation of Non-anticoagulant Heparin Carrier By Glycine Derivatization 
The anticoagulant activity of heparin can be reduced to almost negligible 
activity by derivatizing its carboxylate groups with glycine residues as 
reported Danishefsky et al. (1971); Danishefsky et al. (1972)!. This 
non-anticoagulant heparin (Nac-heparin) can then be utilized as a modified 
glycosaminoglycan carrier. According to one present method of glycine 
conjugation, 0.75 g of heparin is weighed into a 100 mL beaker and 
dissolved in 25 mL of pharmaceutical-grade water. Glycine, 0.75 g, is 
added and the pH of the resulting solution adjusted to 4.75 with 0.10N 
HCl. 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide HCl (EDC), 0.75 g, is 
weighed into a separate vial, solubilized by adding a minimum amount of 
water, and the pH adjusted to 4.75 with 0.10M HCl. Aliquots of the EDC 
solution are added to the mixture of glycine-glycosaminoglycan over a one 
hour period. After each addition of EDC, the pH is adjusted to maintained 
it at 4.75. After addition of all EDC, the reaction is allowed to proceed 
for an additional two hours with constant stirring and periodic pH 
adjustment. The glycine-heparin conjugate (Gly-HEP) is then precipitated 
by addition of 3 volumes of absolute ethanol. The precipitate is collected 
by centrifugation at 4500 rpm (.apprxeq.2500.times.g) for 15 minutes; and 
washed three times with 20-mL aliquots of ethanol with re-centrifugation. 
EXAMPLE 8 
Preparation of Paired-ion Agents of Ferrioxamine Bound To 
Glycosaminoglycans, Modified and Derivatized Glycosaminoglycans of: 
heparan sulfate, non-anticoagulant heparin oversulfated dermatan sulfate 
chondroitin sulfate, oversulfated chondroitin sulfate and the bacterial 
Sulfatoid, pentosan polysulfate 
Ferrioxamine paired-ion agents are prepared with various glycosaminoglycan 
carriers by mixing appropriate ratios of water solutions of ferrioxamine 
(as in Example 5, above) with the following glycosaminoglycans: (a) 
heparan sulfate of MN=8,500 daltons; (b) non-anticoagulant heparin SPL, ++ 
of MN=10,500 daltons; (c) oversulfated dermatan sulfate of MN=19,000 
daltons; (d) chondroitin sulfate of MN=23,400 daltons; (e) oversulfated 
chondroitin sulfate of MN=14,000 daltons; and (f) pentosan polysulfate of 
MN=2,000 daltons. The ratios of ferrioxamine to glycosaminoglycan and 
sulfatoid carriers are prepared to give a payload of 77.5:22.5% (w/w) of 
ferrioxamine to carrier! (adjusted) by a scaling factor of (mEq 
sulfates/mg of carrier as above)/(mEq sulfates/mg of beef lung heparin*)!. 
Using 0.1 to 1.0N NaOH, the pH of the mixture is adjusted to between 5.5 
and 8, the mixture is stirred continuously for 0.5 to 72 hours and the pH 
re-adjusted between 5.5 and 8. This ferrioxamine:heparin mixture is passed 
through a 0.22 .mu.m filter to remove any residual insoluble iron 
oxides-hydroxides and render the liquid agent aseptic. The aseptic agent 
is stored at 4.degree. C. As indicated, further processing is carried out 
by filling the aseptic liquid in glass vials, followed by freezing and 
lyophilizing, to render the agent as an aseptic lyophilized powder. The 
lyophilized vials are reconstituted by adding sterile water and hand 
mixing for 1 to 5 minutes, to give a reconstituted liquid of desired 
concentration which is ready for injection. The resulting concentrations 
of ferrioxamine and heparin are measured and vial quantities confirmed by 
standard reverse-phase HPLC and macromolecular size exclusion HPLC 
methods, respectively. 
FNT * For beef lung heparin, mEq SO.sub.3.sup.- /g carrier=4.4. 
Although not prepared in the present application, it is apparent that by 
combining the teaching of the present Example with those of previous 
disclosures 07/880,660, 07/803,595, and 07/642,033, ferrioxamine complexes 
can be similarly prepared with additional acidic saccharides, including 
sucrose octasulfate and sulfated cyclodextrins; with additional 
glycosaminoglycans, including keratan sulfate and hyaluronate; and with 
additional sulfatoids, including the bacterial sulfatoid, dextran sulfate. 
EXAMPLE 9 
Preparation of Paired-ion Agents of Gd(III):DTPA-Lys Bound to Dermatan 
Sulfate Carrier 
Gd(III):DTPA-Lys:Dermatan Sulfate paired-ion agents are prepared by mixing 
the water solutions of Gd(III):DTPA-Lys with dermatan sulfate of modal MW 
between approximately 5,000 daltons and 45,000 daltons (as in Example 5, 
above), and in particular, dermatan sulfate of MN=18,000 (Opocrin, S.p.A., 
Modena, Italy), to form a final solution ratio of 77:30% (w/w) of the 
Gd(III):DTPA-Lys active to the Dermatan Sulfate carrier. Several stable 
Agent variations of the resulting liquid have been prepared, wherein the 
concentration of Gd(III):DTPA-Lys ranges from 0.166 to 0.415 mmol/mL, and 
the respective concentration of dermatan sulfate ranges from 35 to 87.5 
mg/ml. 
EXAMPLE 10 
Preparation of a Basic Iron-porphine Chelate; and Paired-ion Binding to 
Heparin 
The soluble, tetra-basic porphine, 
5,10,15,20-tetrakis(1-methyl-4-pyridyl)-21H-23-Hporphine, 40 mg as the 
tetra-p-tosylate salt, is refluxed with Fe(II) chloride, 30 mg, for 2 
hours in 20 mL of dimethylformamide. Evidence of iron complexation is 
observed in the form of a red to dark green color. Solvent was removed by 
evaporation, the solid product dissolved in water. The pH is adjusted to 
7.5 to insolubilize excess ferric iron, followed by filtration of the 
iron-porphine product. A 2 mg/mL solution of iron-porphine complex and ca. 
100% product yield is confirmed by inductively coupled plasma atomic 
absorption. A comparable reaction in water gives ca. 70% yield. 
This iron-porphine complex is added to beef lung heparin dissolved in 
water, ca. 8 Kd, at ratios ranging from 1:20 to 20:1 
(iron-porphine:heparin). This resulted in clear solutions without 
precipitates. Binding of iron-porphine to heparin is nearly 100% as 
evaluated by dialysis against water for 16 hours, using bags with 
molecular weight cutoffs of 3.5 Kd and 12 Kd. Iron-porphine alone is 
nearly completely dialyzed. UV-Visible spectrophotometric titration 
indicates maximum binding occurs at a molar ratio of 18:1 
(iron-porphine:heparin). Since the beef lung heparin used is known to have 
approximately 18 available strongly acidic (sulfate) groups per mole (and 
per heparin chain), these results indicate strong ionic interaction and 
stable (to dialysis) binding of the basic tetraamine porphine complex to 
the sulfate groups of heparin. 
EXAMPLE 11 
Preparation of a Basic Triethylenetetraamine-iron Chelate; and Paired-ion 
Binding to Heparin and Sucrose Octasulfate 
Soluble complexes of triethylenetetraamine and iron(III) are formed by 
dissolving 1.0 g of triethylenetetraamine.2HCl (Syprine.TM.) (Merck, West 
Point, Pa.) in water and adding a 1:1 mole ratio of iron chloride under 
acidic conditions (pH=2) to give a clear yellow solution. Using 0.1N NaOH, 
the pH is adjusted to 6.8, giving a red solution indicative of iron 
complexation. This solution develops a feathery red precipitate, 
indicative of intermolecular aggregation of the iron-triethylenetetraamine 
complex. 
(a) To this resulting aqueous dispersion of complex is added beef lung 
heparin, to give final complex-to-heparin ratios of between 95:5 and 5:95 
(by weight). At a ratio of 65:35 (complex:heparin) and higher ratios of 
heparin, heparin completely solubilizes the complex. This apparent 
solubilization is indicative of paired-ion binding between 
triethylenetetraamine-iron and heparin. 
(b) To the aqueous dispersion of triethylenetetraamine-iron complex is 
added sucrose octasulfate (SOS), to give final complex-to-SOS ratios of 
between 95:5 and 5:95 (by weight). At a ratio of 65:35 (complex:SOS) and 
higher ratios of SOS, SOS causes the dispersion to become very much finer, 
indicative of paired-ion binding between triethylenetetraamine-iron 
complex and SOS. The absence of complete clarification of this SOS 
paired-ion system relative to that with heparin (above), is due to the 
much higher density of sulfates on SOS relative to heparin, which confers 
substantially increased intermolecular hydrogen bonding on the SOS system. 
Although not directly exemplified, it will be apparent that polyamines with 
the homologous series C.sub.x H.sub.x+y N.sub.x-z, which also form stable 
complexes with Iron(III), can also be used in place of 
triethylenetetraamine-iron complex and SOS in the present invention. 
Preparation of Covalent Conjugates of Deferoxamine Glycosaminoglycan 
Carriers 
Substrates with electrophilic amine groups may be covalently conjugated 
reagents to nucleophilic carboxylate groups of acidic carriers, acidic 
saccharides and acidic glycosaminoglycans as reported Danishefsky et al. 
(1971); Danishefsky et al. (1972); Janoki et al. 1983); Axen (1974); 
Bartling et al. (1974); Lin et al. (1975)!. The coupling reagents 
described in these references activate carboxylate groups toward 
nucleophilic attack. The mechanism involves formation of an activated 
intermediate resulting from reaction of the coupling reagent with the 
carboxylate residues on the carrier. The intermediate undergoes 
nucleophilic attack, typically by an amine functional group. This results 
in formation of a stable covalent conjugate, typically via an amide bond 
between the active and the carrier. Examples 12, 13, and 14 (below) 
describe the synthesis of ferrioxamine-heparin covalent conjugates, 
wherein the ferrioxamine is covalently bound to heparin via three 
different coupling reagents. 
EXAMPLE 12 
Preparation of a Covalent Ferrioxamine-Heparin Conjugate by 
1-ethyl-3-(3-dimethylaminopropyl) Carbodiimide (EDC) Linkage 
Aqueous ferrioxamine, 2.0 g, as prepared in Example 1, is adjusted to pH 
4.75 by addition of 0.10M HCl. Beef-lung heparin (Hepar-Kabi-Pharmacia, 
Franklin, Ohio), 0.75 g, is dissolved 5.0 mL of pharmaceutical-grade water 
and added to the ferrioxamine with constant stirring. The pH of the 
resulting solution is readjusted to 4.75 with 0.10M HCl. The water-soluble 
carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide HCl (EDC), 2 
g, is weighed into a scintillation vial, solubilized in a minimum amount 
of water, and the pH adjusted to 4.75 with 0.10M HCl. Aliquots of EDC 
solution are pipetted into the mixture of ferrioxamine-heparin over a one 
hour period. After each addition of EDC the 0.10M HCl is added to maintain 
the pH at 4.75. After addition of all EDC, the reaction is allowed to 
proceed for an additional two hours with constant stirring. The 
ferrioxamine-heparin conjugate is precipitated by addition of 3 volumes of 
absolute ethanol. This precipitate is collected by centrifugation at 4500 
rpm (.apprxeq.2500.times.g) for 15 minutes and washed three times with 20 
mL aliquots of ethanol plus centrifugation. The complex is further 
purified by redissolving in water and reprecipitating with 3 volumes of 
ethanol plus centrifugation. The final product is collected and dried over 
nitrogen. Ferrioxamine derivatization of heparin is confirmed by 
UV-visible absorbance spectroscopy of the ferrioxamine chelate at 430 nm 
and heparin analysis by size-exclusion HPLC chromatography. 
EXAMPLE 13 
Preparation of a Covalent Ferrioxamine-Heparin Conjugate by 
N-Ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) Linkage 
Beef-lung heparin (Hepar-Kabi-Pharmacia, Franklin, Ohio), 0.50 g, is 
weighed into a 3-necked 100 mL round bottom flask fitted with an inlet and 
outlet for N.sub.2 purge. Anhydrous dimethylformamide (DMF), 20 mL, is 
added with constant stirring and the resulting suspension warmed to 
50.degree. C. under a constant flow of nitrogen. A 30 mole excess 
(.apprxeq.463.7 mg) of N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline 
(EEDQ) is added and the resulting suspension stirred at 50.degree. C. for 
3 hours. The activated EEDQ-activated heparin is collected by 
centrifugation at 4500 rpm (.apprxeq.2500.times.g) for 10 minutes. The 
pellet is washed repeatedly with anhydrous DMF and then 3 times with 
acetone. The activated intermediate is dried under a stream of nitrogen. 
An aliquot of ferrioxamine solution containing 766.3 mg of the iron 
complex, as prepared in Example 1, is pipetted into a 50 mL beaker and 
diluted to 25 mL with anhydrous DMF. In a separate 50 mL beaker, a known 
amount of EEDQ-activated heparin is suspended in 50 mL of anhydrous DMF 
with constant stirring. The DMF solution of ferrioxamine is pipetted 
slowly into the EEDQ-heparin suspension over a 5 minute period. The 
resulting suspension is stirred continuously for 3 hours at 40.degree. C. 
After cooling to room temperature, the final product is collected by 
centrifugation, washed three times with anhydrous DMF, washed three times 
with acetone, and dried under nitrogen. Confirmation of conjugate 
formation is performed as in Example 12. 
EXAMPLE 14 
Preparation of a Covalent Ferrioxamine-Heparin Conjugate by 
Carbonyldiimidazole (CDI) Linkage 
An activated intermediate of beef-lung heparin (Hepar-Kabi-Pharmacia, 
Franklin, Ohio) is prepared by weighing 3.0 g of heparin into a 50 mL 
round bottom flask and adding 25 mL of anhydrous dimethylformamide (DMF) 
with constant stirring. Carbonyldiimidazole (CDI), 608.1 mg, (10 mole 
excess relative to heparin) is weighed into a separate vial and dissolved 
in 20 mL of anhydrous DMF. The DMF solution of CDI is added to the 
DMF-heparin suspension and stirred at 30.degree. C. for one hour. The 
CDI-activated heparin is collected by centrifugation, washed repeatedly 
with acetone to remove unreacted CDI and residual DMF, and dried under 
nitrogen. 
The deferoxamine-heparin conjugate is prepared by weighing 1.0 g of the 
CDI-activated heparin into a 50 mL round bottom flask and suspending this 
in 25 mL of anhydrous DMF. Deferoxamine, 250 mg, prepared as in Example 1, 
is weighed into a separate round bottom flask and dissolved in 20 mL of 
anhydrous DMF. The deferoxamine free base solution is added slowly to the 
CDI-heparin suspension and stirred continuously for 16 hours at 75.degree. 
C. The deferoxamine-heparin conjugate is collected by centrifugation at 
4500 rpm (.apprxeq.2500.times.g) for 15 minutes, washed repeatedly with 
anhydrous DMF, washed repeatedly with acetone, and dried under nitrogen. 
The resulting product is dissolved in water, and its concentration 
determined by UV-Visible spectroscopy. A stoichiometric quantity of 
aqueous FeCl.sub.3 is added and the resulting solution adjusted gradually 
to pH 6.5 and stirred for 2 hours. This results in a deep brown-red 
product. This ferrioxamine-heparin conjugate is separated from any 
residual substrates and intermediates by dialysis through a 2,000 MW 
cutoff bag against 150 volumes of water. The retentate is collected and 
concentrated by rotary evaporation. Confirmation of derivatization is 
performed as in Examples 12 and 13. 
EXAMPLE 15 
Preparation of a Covalent Heparin-Diethylenetriaminepentaacetate Conjugate 
(DTPA-heparin) 
DTPA-functionalized carriers are prepared in aqueous media from the 
reaction of diethylenetriaminepentaacetic dianhydride (cDTPAA; 
Calbiochem-Bhering Corp.) and a molecule containing a nucleophilic 
functional group. Beef-lung heparin (Hepar-Kabi-Pharmacia, Franklin, 
Ohio), 1.5 g, is dissolved in 75.0 mL of 0.05M HEPES buffer and the pH 
adjusted to 7.0 with 0.10M NaOH. cDTPAA, 4.5 g (.apprxeq.100 mole excess 
relative to heparin), is weighed out and divided into 20 equal (225 mg) 
aliquots. An aliquot of cDTPAA is added to the heparin solution every 3-5 
minutes until all cDTPAA has been added. The pH of the solution is 
monitored continuously throughout cDTPAA addition and maintained at pH 7.0 
with 0.10M NaOH. After addition of the last aliquot of cDTPAA, the 
solution is stirred for an additional 30 minutes. The DTPA-heparin 
solution is dialyzed through 1000 MW bags against 150 volumes to remove 
non-conjugated DTPA. The resulting conjugate is concentrated by 
nitrogen-evaporation at 37.degree. C. and stored at 4.degree. C. 
EXAMPLE 16 
Preparation of Gadolinium(III) and Iron(III) Chelates of DTPA-heparin 
Covalent Conjugate 
The DTPA-heparin conjugate of Example 15 is further prepared in the form of 
paramagnetic metal chelates of the DTPA group with gadolinium(III) or 
Fe(III), by pipetting the required volume of DTPA-heparin into a 125 mL 
Erlenmeyer flask, adding a 1.5-to-10 mole excess of the paramagnetic metal 
ion oxide, as Gd.sub.2 O.sub.3 or Fe(O)OH, and stirring for 24 to 36 hours 
at 37.degree. C. to obtain solubilization of the metal oxides sufficient 
for complete occupancy of the DTPA groups. The residual metal oxides are 
precipitated by centrifugation at 4500 rpm (.apprxeq.2500 g), and the 
product separated from unreacted metal oxides by filtration through a 
Millipore 0.22 .mu.m GV-type filter, followed by dialysis against 150 
volumes. The concentrations of chelated metal ion and heparin are 
determined by inductively coupled plasma (ICP) and size-exclusion HPLC, 
respectively. In the case of Gd(III), stoichiometric chelation is also 
confirmed by standard xylenol orange titration Lyle et al. (1963)!. 
EXAMPLE 17 
Toxicity Studies of Ferrioxamine:Dermatan Sulfate 
Acute intravenous Toxicity Studies with 14-day recovery and necropsy are 
performed in male and female rats and male and female dogs. At standard 
i.v. injection rates of 0.075 mmol/Kg/min., significant signs generally 
occur only after 5-12.5 1 times the effective imaging dose of 0.155 
mmol/Kg. The LD50 is much greater than 4.5 mmol/Kg and is limited by 
technical aspects of tail-vein infusion. At this rate, some rats can be 
infused with 10 mmol/Kg without untoward effects. At an artificially 
accelerated i.v. injection rate of 0.080 mmol/Kg, deaths in rats can be 
obtained, and the LD50 is between 2.5 and 3.0 mmol/Kg. Terminal necropsy 
reveals no abnormalities in any rats after i.v. injection of 2.2, 3.0 and 
4.5 mmol/Kg (n=5 males and 6 females per dose level). 
A pyramid acute i.v. toxicity study is performed in dogs at escalating 
doses of 0.5, 1.2 and 2.25 mmol/Kg and an infusion rate of 0.012 
mmol/Kg/min in protocol studies. An acute symptom complex of hypotension 
can be obtained, which is minimal and reversible. No deaths occurred and 
terminal necropsy at 14 days revealed no abnormalities (n=2 males and 2 
females, all administered each of the three dose levels, with a 72-hour 
rest interval). 
EXAMPLE 18 
Ferrioxamine:Dermatan Sulfate Selective Contrast Agent: MRI Imaging of 
Lactating Breast Adenocarcinomas in Syngeneic Fisher 344 Female Rats 
As shown in FIGS. 2A-4d, T1-weighted MRI images (TR/TE-800/45 and 550/23) 
are performed at 1.0 and 1.5 Tesla, before (Pre) and after (Post) 
intravenous (i.v.) injection of Ferrioxamine:Dermatan Sulfate Selective 
Paramagnetic Contrast Agent (Example 5), at a Ferrioxamine dose of 0.155 
mmol/Kg into Fisher 344 female rats, with syngeneic breast adenocarcinomas 
inoculated by trocar into the livers, such that tumor diameters at the 
time of imaging are between 1.0 cm and 2.5 cm. Tumors are not conspicuous 
on standard T1-weighted Precontrast images. Following injection of 
Ferrioxamine:Dermatan Sulfate Agent, the tumors (a) become rapidly and 
markedly enhanced at an early postinjection time (7 mins) (FIGS. 2A-B); 
(b) display very sharp tumor boundaries against surrounding liver (FIGS. 
2A-B and 4A-D), and discretely demarcated, darker central region of tumor 
necrosis (FIGS. 2A-B) (allowing tumor perfusion and function to be 
spatially resolved and assessed within different, very small anatomical 
subregions); (c) exhibit sustained contrast for longer than 64 minutes 
postinjection (MPI) (FIGS. 4A-D, MRI images; FIG. 5, quantitative 
region-of-interest, ROI, analysis) with continued very well defined tumor 
borders at prolonged imaging intervals. MRI images and microwave augmented 
iron stains of the freshly excised, 7 MPI tumors, indicate that tumor-site 
localization of the Ferrioxamine active occurs only when it is bound 
(non-covalently) to carrier (FIGS. 2A-B and 4A-D) and not when 
administered in free form (Active alone) (FIGS. 3A-B). As shown in FIGS. 
8A-C, lung metastases of the liver tumor are rapidly and sensitively 
enhanced in very small 2-mm to 3-mm nodules at an early post-contrast 
interval; and this enhancement of the tumor at lung sites is also 
sustained for a prolonged period with high sensitivity plus retention of 
very sharp tumor boundaries against normal lung. The sustained intervals 
shown in FIGS. 8A-C are much longer than those typically reported for 
Gd:DTPA dimeglumine contrast enhancement at body organ sites. 
EXAMPLE 19 
Ferrioxamine:Dermatan Sulfate Selective Contrast Agent: MRI Imaging of 
Prostate AT-1 Carcinomas in Syngeneic Copenhagen Rats and Comparison with 
Gd(III)DTPA 
As shown in FIGS. 9A-E and 10A-E, T1-weighted MRI images (TR/TE-250/80) 
performed at 4.7 Tesla, before (Pre) and after (Post) intravenous (i.v.) 
injection of Ferrioxamine:Dermatan Sulfate Selective Paramagnetic Contrast 
Agent prepared as in Examples 2 and 5, and injected i.v. at an Iron(III) 
dose of 0.155 mmol/Kg (FIGS. 9A-E); compared to Gadolinium DTPA 
dimeglumine, injected i.v. at a Gd(III) dose of 0.100 mmol/Kg (FIGS. 
10A-E); each of these agents being administered to Copenhagen rats with 
syngeneic AT-1 prostate adenocarcinomas inoculated into previously 
prepared skin pouches Hahn, et al. !, such that tumor diameters at the 
time of imaging are between 1.0 cm and 2.5 cm. Ferrioxamine:Dermatan 
Sulfate produces a rapid large enhancement of the Outer Rim of tumor and 
also of the Vascular Array which fans out from the tumor pedicle which 
carries a high majority of the tumor vasculature. Sustained contrast and 
delineation of these elements remains present through kinetic time points 
of 40 minutes. By comparison, following Gd:DTPA dimeglumine, the outer rim 
is not well delineated, even at the earliest post-contrast interval (7 
MPI). Marked early contrast fading occurs overall in the tumor at 20 MPI, 
and some agent sequesters in the central, poorly perfused (cystic) regions 
of tumor (as is typically reported for Gd:DTPA when used for imaging at 
body sites). At 40 MPI, enhancement reverts to essentially background 
levels, and at 60 MPI, there is no residual contrast, except for central 
cystic regions. 
EXAMPLE 20 
MRI Contrast Enhancement of Acute Dog Myocardial Infarcts by 
Ferrioxamine:Dermatan Sulfate 
As shown in FIGS. 11A-D, T1-weighted MRI ECG-gated cardiovascular images 
are performed at 0.5 Tesla, before (Pre) and after (Post) rapid 
intravenous (i.v.) infusion of Ferrioxamine:Dermatan Sulfate Selective 
Paramagnetic Contrast Agent injected i.v. at an Iron(III) dose of 0.155 
mmol/Kg into German Shepherd dogs with acute, 90-min myocardial infarcts 
(ligature of proximal left anterior descending coronary artery) followed 
by reperfusion for ca. 90 minutes prior to contrast agent infusion. At 7 
MPI, Ferrioxamine:Dermatan gives strong enhancement of the infarct zone, 
and in particular distinguishes the outer boundary of the infarct, which 
represents the putative marginal zone of the infarct amenable to potential 
recovery, from the central darker region, which represents the putative 
irreversible central infarct. Sustained strong enhancement and zonal 
demarcation is present through 40 MPI. Ferrioxamine injected without 
carrier at 0.155 mmol/Kg, gives no detectible enhancement. In these 
studies, infarct sizes and positions are documented by double dye infusion 
performed immediately after MRI imaging. 
EXAMPLE 21 
Comparison of MRI Tumor-imaging Potency In Vivo with Ferrioxamine Active 
Bound to Various Sulfated Glycosaminoglycans 
Based on low anticoagulant activity, safety and projected site-localization 
potential, certain alternative glycosaminoglycan carriers and certain 
alternative physical forms of the resulting Selective MRI Contrast Agents 
are compared for their relative in vivo potencies of carrier-mediated 
tumor localization of bound Ferrioxamine. Because of its high spatial 
resolution and capacity to detect subtle quantitative differences in agent 
localization, the AT-1 prostate tumor model of Example 19 is used. 
TABLE 2 
______________________________________ 
Concentra- 
Relative 
Form tion Potency 
FIG. (Liquid/ (metal, (scale of 
NO. Agent Lyo) mmol/mL) 
1-6) 
______________________________________ 
12A-D Ferrioxamine 
Lyo 0.415 3.5 
Dermatan-SO.sub.3.sup.- 
13A-D Gd:DTPA-Lys Liquid 0.415 6 
Dermatan-SO.sub.3.sup.- 
14A-D Ferrioxamine 
Lyo 0.332 4.5 
Oversulfated 
Dermatan-SO.sub.3.sup.- 
15A-D Ferrioxamine 
Lyo 0.332 5 
Oversulfated 
Chondroitin-SO.sub.3.sup.- 
16A-D Ferrioxamine 
Lyo 0.332 3.5 
Heparan Sulfate 
______________________________________ 
Carriers of shorter chain length than the glycosaminoglycans, namely 
pentosan polysulfate, are found to be less potent (typically only 2/6 on 
the scale above) and remain at the tumor site for intervals of less than 
about 20 minutes, whereas the GAGs shown in the table above, are much more 
potent and have considerably longer tumor site localization intervals. In 
comparing these carriers, there is a slight-to-moderate trend towards 
increased carrier potency based on carrier sulfate charge density. 
Lyo=Lyophilized powder form 
SO.sub.3.sup.- =Sulfate (e.g. SO.sub.3.sup.- =dermatan sulfate) 
While the compositions and methods of this invention have been described in 
terms of preferred embodiments, it will be apparent to those of skill in 
the art that variations may be applied to the composition, methods and in 
the steps or in the sequence of steps of the method described herein 
without departing from the concept, spirit and scope of the invention. 
More specifically, it will be apparent that certain agents which are both 
chemically and physiologically related may be substituted for the agents 
described herein while the same or similar results would be achieved. All 
such similar substitutes and modifications apparent to those skilled in 
the art are deemed to be within the spirit, scope and concept of the 
invention as defined by the appended claims. 
The following references are incorporated in pertinent part by reference 
herein for the reasons cited above. 
REFERENCES 
Axen (1974) Prostaglandins, 5(1):45. 
Bacchi et al. (1993) Am. J. Pathol., 142:579. 
Bartling et al. (1974) Biotechnology and Bioengineering, 16:1425. 
Bevilacqua et al. (1993) J. Clin. Invest., 91:91. 
Bevilaqua et al. (Jul. 10, 1993) Congress of Intl. Soc. of Thrombosis and 
Hemostasis. 
Boneu et al. (1992) Heparin and Related Polysaccharides, Plenum Press, NY, 
pg. 237. 
Brechbiel et al. (1986), Inorg. Chem., Vol 25, p. 2772-2781. 
Crumbliss et al. (1991), HANDBOOK OF MICROBIAL IRON CHELATES, Chapter 7, 
CRC Press. 
Danishefsky et al. (1972) Thrombosis Research, 1:173. 
Danishefsky et al. (1971) Carbohydrate Research, 16:199. 
Elices et al. (1990) Cell, 60:577-584. 
Geraldes et al. (1985) Proc. Soc. Mag. Res. Med., Vol. 2, p. 860. 
Hahn et al. (1993) Mag. Res. Imaging, 11:1007. 
Hashimoto et al. (1983) J. Nucl. Med. Vol. 24, p. 123. 
Huber et al. (1991) Science, 254:99-102. 
Janoki et al. 1983) Int. J. Appl. Radiat. Isot., 34(6):871. 
Kjellen et al. (1977) Biochem. and Biophys. Res. Comm., 74:126-133. 
Levine (1993) FASEB Journal, 7:1242. 
Li et al. (1993) Bioconjugate Chem. Vol. 4, p. 275-283. 
Lin et al. (1975) Analytical Biochemistry, 63:485. 
Lindahl and Hook (1978) Ann. Rev. Biochem., 47:385. 
Lorant et al. (1993) J. Clin. Invest., 92:559. 
Lyle et al. (1963) Talanta, 10:1177. 
Montrucchio et al. (1993) Am. J. Pathol., 142:471. 
Munro et al. (1992) Am. J. Pathol., 141:1397. 
Nikkari et al. (1993) Am. J. Pathol., 143:1019. 
Ramirez et al. (1973) J. Macromol. Sci-Chem., A7(5):1035. 
Ranney (1990) U.S. Pat. No. 4,925,678. 
Ranney (1992) U.S. Pat. No. 5,108,759. 
Ranney, patent application Ser. No. 07/642,033. 
Ranney, patent application Ser. No. 07/803,595. 
Ranney, patent application Ser. No. 07/880,660. 
Ransohoff et al. (1993) FASEB Journal, 7:592. 
Rice et al. (1991) Am. J. Pathol., 138:385. 
Sasseville et al. (1992) Am. J. Pathol., 141:1021. 
Sessler et al., U.S. Pat. No. 5,159,065. 
Sessler et al., U.S. Pat. No. 5,252,720. 
Sessler et al., U.S. Pat. No. 4,935,498. 
Sharon et al. (January 1993) Scientific American, pg. 83. 
Steinhoff et al. (1993) Am. J. Pathol. 142:481. 
Strieter et al. (1992) Am. J. Pathol., 141:1279. 
Travis (1993) Science, 260:906.