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WO1996019242A1 - Complexes de sulfate de dermatan et de medicaments, ameliorant la pharmacocinetique - Google Patents

Complexes de sulfate de dermatan et de medicaments, ameliorant la pharmacocinetique Download PDF

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Publication number
WO1996019242A1
WO1996019242A1 PCT/US1994/014776 US9414776W WO9619242A1 WO 1996019242 A1 WO1996019242 A1 WO 1996019242A1 US 9414776 W US9414776 W US 9414776W WO 9619242 A1 WO9619242 A1 WO 9619242A1
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Prior art keywords
drug
dermatan sulfate
tumor
carrier composition
drug carrier
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PCT/US1994/014776
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English (en)
Inventor
David F. Ranney
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Access Pharmaceuticals, Inc.
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Publication date
Application filed by Access Pharmaceuticals, Inc. filed Critical Access Pharmaceuticals, Inc.
Priority to PCT/US1994/014776 priority Critical patent/WO1996019242A1/fr
Priority to EP95907242A priority patent/EP0794796A1/fr
Priority to JP8519745A priority patent/JPH10510831A/ja
Priority to AU15537/95A priority patent/AU709008B2/en
Publication of WO1996019242A1 publication Critical patent/WO1996019242A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/61—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule the organic macromolecular compound being a polysaccharide or a derivative thereof

Definitions

  • Such a form of drugs is necessary in order to protect the normal vascular endothelium, organs, tissue cells from the toxic effects of drugs, protect the drug from endothelial and tissue metabolism during transit, and make the drug bioavailable at a controlled therapeutic rate selectively within the target tissues and tissue lesions, including solid tumors.
  • sulfates which bind multiply to receptors or antigens which are either synthesized by disease-induced endothelium or are synthesized at other sites but become selectively bound to the induced endothelial receptors at sites of disease. (Ranney, Biochem. Pharmacology, V. 35, No. 7, pp. 1063-1069 (1986)) .
  • the present invention describes improved novel compositions, carriers, 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 in vivo selective drug localization, accumulation and action at sites of disease, including but not limited to solid tumors.
  • 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 drugs and metal chelates at appropriate ratios with anionic or acidic saccharide carriers, thereby forming strong solution-state and dry-state paired-ion complexes and salts, respectively, 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) and in particular, dermatan sulfates. 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.
  • oligomeric and polymeric, hydrophilic and substantially completely hydrophilic carrier substances are included among the preferred carriers for agents to be used for treating tumors, cardiovascular infarcts and other types of local disease.
  • amphoteric and hydrophobic carriers may be favored for certain therapeutic applications.
  • Drugs and metal chelators most useful in this invention include those which contain cationic, basic and basic-amine groups for binding to the carrier, and which are effective to treat local disease conditions either directly or indirectly, including by chelation of 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.
  • 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 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
  • 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.
  • 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 are 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.
  • 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 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 carriers comprising natural 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 comprise essentially the carbohydrate portions of cell-surface and tissue matrix proteoglycans.
  • proteoglycans 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.
  • 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 parenchy al 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 (d) 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.
  • 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, magne ⁇ iurr, mangar.er*'- , iron, gadolinium and aluminum ions.
  • NAC non-
  • a special class c i such solution complexes and salts includes those strong complexes and salts formed by electrostatic or paired-ior. 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.
  • sulface 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.
  • dermatan sulfates with a native sulfate/carboxylate ratio preferably in the range of from 0.7:1 to 1.8:1, more preferably between 0.9:1 and 1.5:1 and typically 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
  • the dermatan sulfate class of glycosaminoglycans and especially the new special class of dermatan sulfates which contain selectively oversulfated oligosaccharide sequences, have the further unique advantages of higher potency combined with very low toxicity as carrier substances of associated or bound actives (i.e., dermatan sulfate-actives, DS-actives) .
  • the dermatan sulfates show these surprising and unexpected advantages over other glycosaminoglycan polysulfates, with S0 3 -/COO- ratios in the range of between 2:1 and 3.7:1 and sulfur contents of greater than or equal to 10% (weight basis -- indicative of their much higher sulfate contents) .
  • the new special class of dermatan sulfates (as described at length below) , which is enriched for selectively oversulfated oligosaccharide sequences without comprising oversulfated or polysulfated molecules overall throughout the entire chain length (the latter being characterized by SO 3 -/COO- ratios greater than or equal to 2.0:1 and sulfur contents greater than or equal 10%) , have the further surprising and unexpected advantage of more strongly binding to the selectively induced receptors of endothelium, tissue matrix and target-cells at sites of disease (including tumors) by means of the complementary, selectively oversulfated oligosaccharide sequences of these new special dermatan sulfates.
  • these new special dermatan sulfates exhibit surprisingly and unexpectedly more potent site localization and site- targeting potencies than would otherwise be expected based on their moderately low overall S0 3 -/COO- ratio and sulfation and on their related extremely low cellular and systemic toxicity properties and side-effect profiles.
  • derivatization of the acidic saccharide and glycosaminoglycan carriers may be accompanied by the basic metal chelator itself.
  • 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.
  • 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.
  • 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.
  • covalent active-carrier agent compositions of low substitution ratio may be useful for certain in vivo applications of typically narrow range
  • non- covalent active-carrier agent compositions of high substitution ratio may be useful for other in vivo applications of typically broader range.
  • non-covalent agents may be particularly useful for the majority of diagnostic imaging applications and for most therapeutic applications, wherein 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 drug toxicity.
  • compositions represent additional substantial improvements over prior, non-selective and covalently conjugated active-carrier agents.
  • the resulting agents are broadly useful for: (a) site-selective drug localization, including tumors, infections and cardiovascular disease with an acute endothelial induction; (b) MRI contrast and spectral enhancement, Ultrasound contrast enhancement, and X-Ray contrast enhancement, where relatively high administered doses may be favored or required; (c) Nuclear Medical or Radionuclide imaging and therapy, where enhanced clearance of the non-targeted dose may be favored or required: and (d) certain high-dose, extended-release or sustained-effect therapy may be favored cr required.
  • Such therapeutic agents include but are n t li ⁇ ted tc those useful at a broad variety of crgan sites and medical indications, for the treatment cf : ⁇ a) acute vascular ischemia, acute infarct, acute vascular damage, shock, hypotension, restenosis, tumors and tumor angiogenesis and parenchy al-cell 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 and drug therapy are important indications for which high payload and controlled release of active agents are important unique advantages in addition to site selective localization (see below) .
  • potentially therapeutic metal ions generally useful for trans chelation at sites of disease may include divalent and trivalent cations selected from the group consisting of: iron, manganese, chromium, copper, aluminum, nickel, gallium, indium, 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 consisting of: phosphorous, sulfur, gallium, iodine, germanium, cobalt, calcium, rubidium, yttrium, technetium, ruthenium, rhenium, indium, tin, iridium, platinum, thallium, strontium 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.
  • 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 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 consisting of: calcium, manganese, magnesium, chromium, copper, zinc, nickel, iron, gallium, indium, 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 consisting of magnesium, manganese, chromium, zinc and calcium, iron, copper and aluminum. It will be obvious to those skilled in the art that various ones 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.
  • compositions described in this invention give surprising and unexpected improvements of performance and use which include:
  • Diagnostic and drug enhancement can be made to occur by a number of mechanisms, the principal ones being:
  • 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.
  • Acidic or anionic saccharides including glycosaminoglycans, dermatan sulfates and the new special dermatan sulfates, localize at target sites by binding first to complementary receptors on disease-site vascular endothelium, induce very rapid (ca.
  • the new special class cf dermatan sulfates appears to perform this complementary rinding function via their selectively enriched oversulfated saccharide sequences, which correlate with an enriched heparin cofactor II activity of at least about 220 U/mg, and which appear to bind the positively charged, cationic and/or structurally complementary receptors or lectins that are selectively induced on disease-site endothelium, tissue matrix and target cells (including in tumors) .
  • these binding and targeting functions and potencies occur without either the overall high sulfation/polysulfation or the incumbent toxicity and safety disadvantages thereof (as otherwise described herein) .
  • 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.
  • acidic saccharide and sulfated 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.
  • 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.
  • mediators include cytokines, chemoattractants, cytotoxins, induced cell-surface adhesions, selections 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.
  • 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.
  • alien mediators such as bacterial lipopolysaccharides (LPS) , viral RNA, and tumor-derived inducers, including EMAP II, and chemoattractants may also be released.
  • interleukin 1 tumor necrosis factor
  • TGF tumor necrosis factor
  • VEGF/VPF vascular endothelial growth factor/vascular permeability factor
  • TGF-beta transforming growth factor beta
  • LPS Lipopolysaccharide
  • MCP monocyte chemoattractant protein
  • IL-8 interleukin 8
  • IL-3 interleukin 3
  • IL-6 tumor-derived inducers and chemoattractant peptides (as above)
  • various prostaglandins and thromboxanes include: interleukin 1 (IL-1) , tumor necrosis factor (TNF) , vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) , transforming growth factor beta (TGF-beta) , Lipopolysaccharide (LPS) , single and double stranded nucleotides, various interferons, monocyte chemoattractant protein (MCP) ,
  • 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 including VEGF/VPF and monocyte chemoattractant protein (MCP) ; and 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.
  • MCP monocyte chemoattractant protein
  • the induced endothelial adhesive molecules 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.
  • coagulation factors especially fibronectin, tissue factor, thrombin, fibrinogen, fibrin, and their split products, especially fibronectin split products and fibrinopeptide A
  • platelet-derived factors platelet activating factor (PAF) , glycoprotein Ilb/IIIa complex
  • white-cell a) L-selectin, and (b) integrins, including VLA-4 (very late antigen 4)
  • VLA-4 very late antigen 4
  • vascular endothelial growth factor/vascular permeability factor VEGF/VPF
  • VEGF/VPF vascular endothelial growth factor/vascular permeability factor
  • VEGF/VPF is a chemically basic growth factor which is quite highly selective for endothelial cells versus fibroblasts and other cell types [Senger et al . (1994) ; Nicosia et al . (1994) , incorporated by reference herein] . It appears to be a key growth factor for stimulating the long-term endothelial 5 angiogenesis in many or most human and animal tumors, and in AIDS-associated Kaposi's sarcoma [Connolly et al . (1989) ; Weindel et al . (1992) , both incorporated by reference herein] . In certain instances, VEGF/VPF may also be
  • VEGF/VPF and platelet-derived growth factor, PDGF-BB are reported recently to be the only species of the group of basic, GAG-binding growth factors which have significant angiogenic potency in
  • VEGF/VPF both provides and induces receptor targets for binding of GAG carrier substances in tumors and potentially in other pathologic lesions.
  • tissue chemoattractants including VEGF/VPF, MCP (Yamashiro et al . , 1994) and IL-8, which comprise a family of arginine-rich, 8Kd, heparin-binding proteins reported to bind GAGs/ACs [Huber et al . (1991) , incorporated by reference herein] ;
  • VCAM-1 vascular cell adhesion molecular
  • ICAM-1 inducible cell adhesion molecule
  • vWF von Willebrand's factor
  • 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;
  • 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 selections, and amplify adhesiveness and activation at the initial endothelial P- selectin sites for GAGs/ACs [Elices et al . (1990) ; Lorant et al . (1993)] ;
  • 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 .
  • 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.
  • GAG/AC carriers selectively bind the induced and exposed determinants listed in Nos. 1-7,
  • the carrier substance has ultivalent 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;
  • Controlled release of the diagnostic or drug activity from carriers comprising GAGs/ACs occurs gradually within the diseased site, thereby resulting in targeted controlled release;
  • 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 prostatic adenocarcinomas; [Kjellen et al . (1977)] 13.
  • the carriers are hydrophilic or essentially completely hydrophilic, these carriers cause their bound actives to migrate (permeate) deeply into and throughout the tumor mass even at microanatomic sites distant from the tumor's typically irregularly spaced microvessels; and also 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 and/or their associated active substances (diagnostics or therapeutics) undergo selective uptake (internalization) by abnormal cells within tissue site and preferentially avoid uptake by normal cells within the site, thereby giving:
  • 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;
  • the tumor-selective GAG- binding cytokines are now known to be present in all three of the following microanatomic locations: tumor-cell surface, tumor extracellular matrix, and local tumor neovascular endothelium.
  • these cytokines provide receptor targets for GAG-agents at all three of the key tumor sites: tumor endothelium, tumor extracellular matrix, and tumor cells proper.
  • the presence of these cytokines selectively on tumor endothelium allows for site-selective binding of intravascularly administered GAG-agents to tumor microvessels and very rapid (ca.
  • VEGF/VPF and MCP cytokines may account for selective tumor-cell internalization of GAG-agents, as shown in certain of the Examples below.
  • the presence of these cytokines plus the GAG-binding peptides of No. 6 (above) in the large extracellular volumes of the tumor matrix accounts in part, for the large tumor-tissue reservoirs of GAG-associated agents (including metal chelates) which are observed by MRI contrast enhancement (see Examples below) .
  • GAG-associated agents including metal chelates
  • the combination of: (1) prolonged tumor retention of Gag-agents as an extracellular reservoir (depot) ; (b) tumor-cell internalization of a portion of this extracellular agent; and (c) very rapid blood and body clearance of the non- targeted portion, provides the following surprising and unexpected advantages for in vivo imaging (including MRI contrast enhancement) and therapy: (a) enhanced tumor selectivity; (b) prolonged, high "areas under the curve" (AUC's) in tumor; (c) short, low AUC's in blood; (d) minimization of local and systemic toxicities.
  • MCP Experimental autoimmune encephalomyelitis in mice [Ransohoff et al . (1993)] ;
  • IL-8 Neovascularization: [Strieter et al . (1992)] ;
  • VCAM-1 VCAM-1
  • INCAM-110 Chronic inflammatory diseases, including sarcoidosis [Rice et al . (1991)] .
  • Integrin, beta 1 subunit cell adhesion receptor inflammatory joint synovium [Nikkari et al . (1993) ] .
  • sialyl Lewis x 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:
  • 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.
  • GAG molecular weights of generally ca. 8,000 to 45,000 MW, preferably 10,000 to 23,000 MW and more preferably 13,000 to 19,000 MW, are important in order to:
  • the paramagnetic metal-ion chelates and images obtained therewith are intended to be demonstrative of agent localizations in sites of disease, including in tumor sites, and to be generally reflective of the disease-site levels, distributions and residence times, as well as of the blood and organ clearance patterns and kinetics, all of which may be useful in interpreting the targeting, localization, accumulation, cellular internalization, and blood and body clearance of therapeutic agents, including agents useful in treating tumors, infection, cardiovascular diseases and other local sites of disease, as described herein.
  • the present invention encompasses novel agents comprising cationic or chemically basic, amphoteric or hydrophobic therapeutic agents, including peptides, polypeptides and proteins, and metal chelators and metal- ion chelates in association with hydrophilic carriers of anionic or chemically acidic saccharides, sulfatoids and glycosaminoglycans.
  • 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.
  • 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- triethylenetetramine, gadolinium DTPA-lysine, gadolinium N-methyl-1, 3-propanediamine (N-MPD) -DTPA, 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, essentially pur
  • 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.
  • MRI magnetic resonance image
  • compositions and methods of the present invention 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.
  • 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.
  • the chelator for metal ions which has a cationic group is bound to an anionic, hydrophilic carrier by non-covalent electrostatic binding.
  • 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.
  • the said chelator may be basic.
  • 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.
  • the chelator has a formation constant for paramagnetic metal ions of at least about 10 14 .
  • 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.
  • 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, samarium, tin and strontium.
  • non-radioactive or radioactive phosphorous, sulfur or iodine may be bound directly to the carrier (below) . 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.
  • 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.
  • the carrier may be, but is not limited to heparin, desulfated heparin, glycine-conjugated heparin, heparan sulfate, dermatan sulfate, essentially purified dermatan sulfate with a sulfur content of up to 9% (w/w) and with selective oligosaccharide oversulfation, hyaluronic acid, pentosan polysulfate, dextran sulfate, sulfated cyclodextrin or sulfated sucrose.
  • the chelator is a chelator of iron ions.
  • the chelator is a hydroxamate, and more preferably it is deferoxamine.
  • the chelator together with the metal ion is ferrichrome, ferrioxamine, enterobactin, ferrimycobactin or ferrichrysin.
  • the chelator is deferoxamine, the carrier is heparin, or a heparin fragment and the agent further comprises iron(III).
  • the chelator is deferoxamine and the carrier is dermatan sulfate or a dermatan sulfate fragment and the agent may further comprise chelated iron(III).
  • the invention may also comprise deferoxamine bound to a carrier selected from the group consisting of heparin, heparan sulfate, dermatan sulfate, essentially purified dermatan sulfate with a sulfur content of up to 9% (w/w) and with selective oligosaccharide oversulfation 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.
  • 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.
  • 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" ,N" ' -tetraacetate (DOTA) .
  • the carrier may also be defined further as being com lement:a y t: endothelial determinants selectively induced at disease sites.
  • 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 l-ethyl-3- (3- dimethylaminopropyl) carbodiimide, N-ethoxycarbonyl-2- ethoxy-1,2-dihydroquinoline, or carbonyldiimidazole.
  • 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 essentially purified dermatan sulfate with a sulfur content of up to 9% (w/w) and with selective oligosaccharide oversulfation.
  • 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, essentially purified dermatan sulfate with a sulfur content of up to 9% (w/w) and with selective oligosaccharide oversulfation, hyaluronic acid, pentosan polysulfate, dextran sulfate, sulfated cyclodextrin or sulfated sucrose.
  • a hydrophilic carrier selected from the group consisting of heparin, desulfated heparin, glycine-conjugated heparin, heparan sulfate,
  • 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 S9 iron or 67 gallium.
  • the invention is an agent for enhancing body imaging, the agent comprising diethylenetriaminepentaacetate-lysine or N-methyl-1, 3-propanediamine DTPA, chelated Gd(III) and a glycosaminoglycan carrier bound to said diethylenetriaminepentaacetate-lysine.
  • 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) .
  • the invention is an agent comprising ferrioxamine bound by non-covalent electrostatic binding to dermatan sulfate or essentially purified dermatan sulfate with a sulfur content of up to 9% (w/w) and with selective oligosaccharide oversulfation.
  • the invention is an agent for enhancing body imaging, including MRI imaging and spectral shift, the agent comprising gadolinium (III) chelated to N-methyl-1,3-propanediamine- diethylenetriaminepentaacetate (N-MPD-DTPA) , the N-MPD- DtPA being bound or in association most preferably by paired-ion or other non-covalent means or alternatively preferably bound by covalent means to a glycosaminoglycan, preferably dermatan sulfate, and most preferably the new special class of dermatan sulfate, and most preferably the new special class of dermatan sulfates containing selectively oversulfated oligosaccharide sequences.
  • gadolinium (III) chelated to N-methyl-1,3-propanediamine- diethylenetriaminepentaacetate (N-MPD-DTPA) N-methyl-1,3-propanediamine- diethylenetriaminepentaacetate
  • 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.
  • 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.
  • 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.
  • 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.
  • the invention includes compositions and methods for delivering, localizing and retaining therapeutic actives selectively to sites of local disease, while clearing the non-targeted dose effectively, rapidly and/or non-toxically from the body, so as to minimize local and systemic toxicities and side effects.
  • therapeutic actives and methods of treatments may be for any type and location of local disease site, provided it has a vascular or other access route, and that it has any form of induced vascular receptors, adhesins, or other signals capable of recognition by the carrier substances described herein.
  • such actives for tumor treatment may include but are not limited to: doxorubicin, adriamycin, taxol, vincristine, vinblastine, bleomycin, idarubicin, epirubicin, and also to amsacrine, azacitidine, dideoxyinosine, dihydro-5-azacytidine, ethanidazole, ethiofos, methotrexate, misonidazole, porfiromycin, pyrazoloacridinek, terephthalamidine, taxotere and other taxane derivatives, topotecan, trimetrexate, N-formyl- met-leu-phe-lys, arginine bradykinin, poly-L-lysine, other chemoattractants, biological response modifiers, cytokines, interferons, lymphokines and other agents useful in treating tumors or neoplastic disease, with any of the above used singly or
  • such actives and methods for treating infection may include but are not limited to: gentamicin, amikacin, tobramycin, and other amine, basic, basic peptide, basic polypeptidic, hydrophobic or amphoteric antibiotics or agents for treating bacterial, fungal, mycobacterial, viral or other microbial or microbiological diseases.
  • the present invention may be described in certain embodiments as a drug carrier composition
  • a drug carrier composition comprising a drug in combination with essentially purified dermatan sulfate with a sulfur content of up to 9% (w/w) and with selective oligosaccharide oversulfation, wherein said composition has a non-embolizing size of less than about 500 nm.
  • the drug may be a chelator.
  • the composition may have a size of less than about 250 nm.
  • the drug carrier composition may also be defined further wherein binding to disease induced endothelia causes the endothelia to totally or partially envelop bound drug carrier composition in less than 10 to 15 minutes, and wherein said essentially purified dermatan sulfate with sulfur content of up to 9% (w/w) and with selective oligosaccharide oversulfation, contains Ido-GalNAc4S0 3 , and further comprises IdoA2S0 3 -GalNAc4S0 3 and IdoAGalNAc4, 6S0 3 .
  • the drug carrier composition of the present invention may also be defined in certain embodiments as being a nanoparticle, and the drug may preferably by an oncotherapeutic drug.
  • the oncotherapeutic drug is preferably selected from the group consisting of adriamycin, doxorubicin, epirubicin, daunorubicin, idarubicin or salts thereof, with doxorubicin being the most preferred.
  • the oncotherapeutic drug may alternatively be selected from the group consisting of bleomycin, taxol, taxotere, vinblastine and vincristine, amsacrine, azacytidine, dideoxyinosine, dihydro-5- azacytidine, ethanidazole, ethiofos, methotrexate, isonizadole, porfiromycin, pyrazoloacridinek, terephthalamidine, taxotere, topotecan, trimetrexate and carboplatin or salts thereof.
  • a particular embodiment of the present invention is a drug carrier composition
  • a drug carrier composition comprising a drug selected from the group consisting of doxorubicin, epirubicin, daunorubicin and idarubicin in combination with essentially purified dermatan sulfate with a sulfur content of up to 9% (w/w) and with selective oligosaccharide oversulfation, wherein said composition has a non-embolizing size of less than about 500 nm, preferably less than 250 nm, most preferably less than Z . nm.
  • Embodiments of the present invention also include drug carrier compositions wherein the drug is an antiinfective (antiviral, antimicrobial, antifungal or antitubercular) drug, with gentamycin, tobramycin or amikacin being preferred, or the drug is a biological response modifier (modifying an endogenous biological response) , preferably a biologically active peptide or polypeptide, and more preferably a white cell chemoattractant, bradykinin or poly-L-lysine.
  • the white cell chemoattractant is preferably N-formyl-met-leu-phe- lys (SEQ ID N0:1) .
  • the drug carrier compositions of the present invention may be further defined as being in a pharmaceutically acceptable solution suitable for intravascular or other parenteral injection, and may be formed by paired-ion charge interactions, amphoteric or hydrophobic interactions between the carrier and drug.
  • the present invention is a method of treating an animal for tumors responsive to an oncotherapeutic agent, the method comprising the steps of preparing a drug carrier composition comprising an oncotherapeutic drug in combination with essentially purified dermatan sulfate with a sulfur content of up to 9% (w/w) and with selective oligosaccharide oversulfation, wherein said carrier has a non-embolizing size of 500 nm or less; containing said drug carrier composition in a pharmaceutically acceptable carrier; and administering the drug carrier composition in the pharmaceutically acceptable carrier to an animal.
  • the drug is in a controlled release form and preferably wherein binding of a sample of said drug carrier composition to disease induced endothelia produces an induction of the endothelia to totally or partially envelop the bound sample in less than 10 to 15 minutes.
  • the drug carrier composition may be administered by selected arterial perfusion, to obtain high-efficiency uptake in proximal target organs, tissues or tissue lesions, or it may be administered intravenously to obtain semiselective, medium-efficiency uptake in tissue lesions located at widely distributed systemic sites.
  • the present invention is also a method of treating an animal for tumors responsive to an oncotherapeutic agent, the method comprising the steps of preparing a drug carrier composition comprising an oncotherapeutic drug in combination with essentially purified dermatan sulfate with a sulfur content of up to 9% (w/w) and with selective oligosaccharide oversulfation, wherein said carrier has a non-embolizing size of 500 nm or less; containing said drug carrier composition in a pharmaceutically acceptable carrier; and administering the drug carrier composition in the pharmaceutically acceptable carrier to an animal; wherein said oncotherapeutic drug is selected from the group consisting of adriamycin, doxorubicin, epirubicin, daunorubicin, idarubicin, bleomycin, taxol, taxotere, vinblastine and vincristine or salts thereof.
  • the invention may be described as a method of treating an animal for tumors responsive to an oncotherapeutic agent, the method comprising the steps of preparing a drug carrier composition comprising an oncotherapeutic drug in combination with essentially purified dermatan sulfate with a sulfur content of up to 9% (w/w) and with selective oligosaccharide oversulfation, wherein said carrier has a non-embolizing size of 500 nm or less; containing said drug carrier composition in a pharmaceutically acceptable carrier; and administering the drug carrier composition in the pharmaceutically acceptable carrier to an animal; wherein said oncotherapeutic drug is selected from the group consisting of amsacrine, azacytidine, dideoxyinosine, dihydro-5-azacytidine, ethanidazole, ethiofos, methotrexate, misonizadole, porfiromycin, pyrazoloacridinek, terephthalamidine, taxotere, topote
  • the binding of a sample of said drug carrier composition to disease induced endothelia may produce an induction of the endothelia to totally or partially envelop the bound sample in less than 10 to 15 minutes, or the drug carrier composition is administered by selected arterial perfusion, to obtain high-efficiency uptake in proximal target organs, tissues or tissue lesions, or the drug carrier composition is intravenously administered to obtain semiselective, medium-efficiency uptake in tissue lesions located at widely distributed systemic sites.
  • 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.
  • Administration of the composition of the present invention may involve any mode of administration resulting in contact of the therapeutic agent with the target tumor, disease site or site of infection. This may include intravenous, intraarterial , intracisternal , intraperitoneal, oral or other administration modes.
  • compositions or formulations of the present invention may be prepared dissolved or dispersed in a pharmaceutically acceptable carrier or diluent or in any other pharmaceutically acceptable form.
  • Such pharmaceutically acceptable formulations will generally comprise an effective amount of the compositions, such as doxorubicin:essentially purified dermatan sulfate in a pharmacological preparation.
  • phrases "pharmaceutically or pharmacologically acceptable” refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a human.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutical active substances is well known in the art . Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.
  • compositions of the present invention may thus be formulated for parenteral administration, such as for intravenous, subcutaneous or intramuscular injection; for oral administration, where the compositions may be formulated into tablets, caplets, or other solids; and the compositions may also be formulated into time release capsules and any other form of pharmaceutical currently used, including cremes, lotions, mouthwashes, inhalents and the like, depending upon location of targeted sites.
  • FIG. IA is a control infrared spectrum of diethylenetriaminetetraacetate (DTPA) substrate (see Example 3) .
  • FIG. IB is a control infrared spectrum of L- lysine.HCl substrate (see Example 3) .
  • FIG. 1C is a control infrared spectrum of a physical mixture of these DTPA and L-lysine.HCl substrates without any chemical covalent linkage of the two substrates (see Example 3) .
  • FIG. ID is the experimental infrared spectrum of L- lysine covalently conjugated to DTPA by l-ethyl-3- (3- dimethylaminopropyl) carbodiimide (EDO linkage (see Example 3) . Note the changes (height, width and loss of splitting) in signature peaks in the range of 1250-1700 wavenumbers, which indicate covalent conjugate formation.
  • Ferrioxamine:Dermatan Sulfate Selective Paramagnetic Contrast Agent prepared as in Examples 2 and 5, and injected i.v. at a Ferrioxamine dose of 0.155 mmol/Kg into Fisher 344 female rats, with syngeneic breast adenocarcinoma inoculated previously into the liver, such that tumor diameters at the time of imaging are between 1.0 cm and 2.5 cm.
  • FIG. 2A Precontrast image of liver (tumor not conspicuous) .
  • FIG. 2B Liver image at 7 min postinjection (MPI) of the Selective Paramagnetic Contrast Agent, Ferrioxamine:Dermatan Sulfate (0.155 mmol/Kg) i.v., showing marked contrast enhancement of tumor in right lobe of liver, very sharp tumor boundaries against surrounding liver, and discretely demarcated darker central region of tumor necrosis -- allowing tumor perfusion and function to be spatially resolved and assessed within different, very small anatomical subregions.
  • MPI Selective Paramagnetic Contrast Agent
  • FIG. 3A Precontrast image of liver (tumor is present but not conspicuous) .
  • FIG. 3B Liver image at 7 MPI of Ferrioxamine Active Alone (without any Dermatan Sulfate Carrier) . Note that acute contrast enhancement is only very slight or nonexistent. This differs markedly from the pronounced tumor enhancement seen in Fig. 2B; and it indicates that binding of the Ferrioxamine active by the Dermatan Sulfate carrier is a requirement for tumor-site localization and tumor uptake of Ferrioxamine active.
  • FIG. 4B Liver image at 21 MPI of Ferrioxamine:Dermatan Sulfate Selective MRI Contrast Agent. Note the marked enhancement of main tumor mass and distinct tumor borders. Also note the small, 2-mm, bright enhancement of tumor metastasis in left lobe of liver. This metastasis is completely non-visualized in the Precontrast Tl images.
  • FIG. 4C Liver image at 30 MPI of
  • Ferrioxamine:Dermatan Sulfate Selective MRI Contrast Agent Note the sustained enhancement of main tumor and metastasis.
  • FIG. 4D Liver image at 42 MPI of Ferrioxamine:Dermatan Sulfate Selective MRI Contrast
  • FIG. 5 Region-of-interest (ROD analyses of MRI image intensities from a tumor animal analogous to that shown in FIG. 4A, FIG. 4B, FIG. 4C and FIG. 4D.
  • Upper line tumor ROI's
  • Lower line liver ROI's
  • time points Precontrast
  • FIG. 6 Special histologic stain (heated ferroferricyanide reaction) of formalin-fixed section of syngeneic breast adenocarcinoma excised from liver inoculation site of Fisher 344 female rats: Outer Tumor Rim 7-10 MPI of Ferrioxamine:Dermatan Sulfate Selective MRI Contrast Agent. Note selective staining for ferrioxamine iron (a) strongly positive on and within tumor endothelium, (b) strongly positive in the subendothelia, (c) moderately positive in the extracellular matrix of tumor, and (d) lightly to moderately positive within tumor intracellular sites.
  • ferrioxamine iron (a) strongly positive on and within tumor endothelium, (b) strongly positive in the subendothelia, (c) moderately positive in the extracellular matrix of tumor, and (d) lightly to moderately positive within tumor intracellular sites.
  • FIG. 7A Same tumor, stain, conditions, and post- contrast time as FIG. 6, except tissue section is taken from Central Tumor, 7-10 MPI of Ferrioxamine-.Dermatan Sulfate Selective MRI Contrast Agent. Significant staining positivity is present at all sites as in FIG. 6.
  • FIG. 7B Identical to FIG. 7A, except a different animal with identical type and site of breast tumor, 7- 10 MPI after i.v. Ferrioxamine Active Alone at a Ferrioxamine dose identical to FIG. 6 and FIG. 7A. Note the complete absence of staining positivity. This correlates directly with the results of MRI imaging with the full Agent (Active bound to Carrier) versus that with Active Alone (Active in free form) -- (refer to FIG. 2A and FIG. 2B versus 3) .
  • FIG. 8B Lung Field of same rat at 12 MPI. Note the marked improvement in sensitivity of tumor detection (conspicuity) due to selective, bright enhancement of the lung metastases. Also note the sharpness of tumor boundaries.
  • FIG. 8C Same Lung Field at 17 MPI -- showing sustained enhancement and sustained sharpness of tumor boundaries.
  • the rapid diffusion rates of Gd:DTPA lead to rapidly fuzzy boundaries at early times; and thereby also decrease the sensitivity of detecting pulmonary metastases.
  • FIG. 9A, FIG. 9B, FIG. 9C, FIG. 9D, FIG. 9E prepared as in Examples 2 and 5, and injected i.v. at an Iron(III) dose of 0.155 mmol/Kg; compared to Gadolinium DTPA dimeglumine (FIG. 10A, FIG. 10B, FIG. IOC, FIG. 10D, FIG. 10E) , injected i.v. at a
  • Gd(III) dose 0.100 mmol/Kg; each of these agents being administered to Copenhagen rats with syngeneic AT-1 prostate adenocarcinoma inoculated into previously prepared skin pouches [Hahn et al . (1993)] , such that tumor diameters at the time of imaging are between 1.0 cm and 2.5 cm.
  • FIG. 9A Precontrast image for Ferrioxamine:Dermatan Sulfate Selective Contrast Agent.
  • FIG. 9B 7 MPI of Ferrioxamine :Dermatan Sulfate, liquid form at a ferrioxamine concentration of 0.166 mmol/mL. Note the strong enhancement of Outer Rim and Vascular array which fans out from the tumor pedicle .
  • FIG. 9C Same as FIG. 9B, except 20 MPI. Note the sustained, discrete enhancement of elements in FIG. 9B.
  • FIG. 9D Same as FIG. 9C, except 40 MPI. Note the sustained contrast and delineation of Outer Rim.
  • FIG. 9E Same as FIG. 9D, except 60 MPI. Note the onset of contrast fading.
  • FIG. 10A Precontrast image for Gd:DTPA dimeglumine Nonselective Contrast Agent .
  • FIG. 10B 7 MPI of Gd:DTPA dimeglumine. Note that the Outer Rim is not well delineated, even at this very early post-contrast interval.
  • FIG. IOC Same as FIG. 10B, except 20 MPI. Note the marked early contrast fading overall, with some agent sequestration seen at the central, poorly perfused (cystic) regions of tumor (as is typically reported for Gd:DTPA when used for imaging at body sites) .
  • FIG. 10D Same as FIG. IOC, except 40 MPI. Note that enhancement is nearly reverted to background levels.
  • FIG. 10E Same as FIG. 10D, except 60 MPI. No residual contrast, except for central cystic regions.
  • FIG. 11A, FIG. 11B, FIG. 11C and FIG. 11D show Tl- weighted MRI ECG-gated cardiovascular images performed at 0.5 Tesla, before (Pre) and after (Post) rapid intravenous (i.v.) infusion 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 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.
  • FIG. 11A Precontrast image
  • FIG. 11B 7 MPI, showing strong enhancement of infarct by Ferrioxamine :Dermatan Sulfate Agent, and in particular delineating the boundary of the infarct -- putatively the boundary of the marginal zone. Note the central darker region -- putatively the irreversible central infarct zone.
  • FIG. 11D 40 MPI, same as FIG. 11C, except filling in of central zone; absence of significant overall contrast fading.
  • FIG. 12A, FIG. 12B, FIG. 12C and FIG. 12D show MRI 4.7 Tesla, Tl-weighted images of Copenhagen rats with the AT-1 prostate tumor model (as in FIG. 9A, FIG. 9B, FIG. 9C, FIG. 9D, FIG. 9E, FIG. 10A, FIG. 10B, FIG. IOC, FIG. 10D and FIG. 10E) , but rats are injected i.v.
  • FIG. 12A Precontrast image for Ferrioxamine :Dermatan Sulfate Selective Contrast Agent.
  • FIG. 12B 7 MPI of Ferrioxamine:Dermatan Sulfate, lyophilized reconstituted to a Fe(III) concentration of 0.415 mmol/mL. Note the very strong enhancement of the entire Outer Rim of tumor.
  • FIG. 12C Same as FIG. 12B, except 20 MPI. Note the sustained, very strong enhancement and delineation of Outer Rim.
  • FIG. 12D Same as FIG. 12C, except 40 MPI. Note the sustained very strong enhancement of Outer Rim with the Central Tumor now also starting to enhance brightly. Also note there is virtually no contrast fading at 40 minutes.
  • FIG. 13A, FIG. 13B, FIG. 13C, FIG. 13D show MRI 4.7 Tesla, Tl-weighted images of Copenhagen rats with the AT- 1 prostate tumor model (as in FIG. 12A, FIG. 12B, FIG. 12C and FIG. 12D) , but rats are injected i.v. with Gd(III) :DTPA-Lys:Dermatan Sulfate Selective Contrast Agent in liquid form pre-concentrated to 0.415 mmol/mL
  • Gd(III) and administered at the usual dose of 0.155 mmol of Gd(III) per Kg.
  • FIG. 13A Precontrast image for Gd (III) :DTPA- Lys:Dermatan Sulfate Selective Contrast Agent.
  • FIG. 13C Same as FIG. 13B, except 20 MPI. Note the sustained, very strong absolute enhancement Outer Rim. Also note additionally strong enhancement of the central vascular array (as differentiated from cystic sequestration) .
  • FIG. 13D Same as FIG. 13C, except 40 MPI. Note sustained enhancement of Outer Rim, with overall enhancement just beginning to fade at 40 minutes, but absolute enhancement remaining as bright or brighter in all regions relative to Ferrioxamine :Dermatan Sulfate.
  • FIG. 14A, FIG. 14B, FIG. 14C and FIG. 14D show MRI 4.7 Tesla, Tl-weighted images of Copenhagen rats with the AT-1 prostate tumor model (as in FIG. 13A, FIG. 13B, FIG. 13C and FIG. 13D) , but rats are injected i.v.
  • the Agent lyophilized and reconstituted with water just prior to administration at a concentration of 0.332 mmol/mL Fe(III) and administered at the usual dose of 0.155 mmol of Fe(III) per Kg.
  • FIG. 14A Precontrast.
  • FIG. 14D 40 MPI. Note the equivalent to slightly greater enhancement of Tumor Rim and greater definition of the vascular array at all times, in relation to
  • FIG. 15A, FIG. 15B, FIG. 15C and FIG. 15D show MRI 4.7 Tesla, Tl-weighted images of Copenhagen rats with the AT-1 prostate tumor model (as in FIG. 13A, FIG. 13B, FIG. 13C and FIG. 13D) , but rats are injected i.v. with Ferrioxamine Selective Contrast Agent, wherein the Active is non-covalently bound to Oversulfated Chondroitin Sulfate, the Agent lyophilized and reconstituted with water just prior to administration at a concentration of 0.332 mmol/mL Fe(III) and administered at the usual dose of 0.155 mmol of Fe(III) per Kg.
  • FIG. 15A Precontrast
  • FIG. 15D 40 MPI. Note the moderately greater enhancement of Tumor Rim and greater definition of the vascular array at 7 MPI, and the only slightly greater enhancement at the two later times, in relation Ferrioxamine bound to Native Dermatan Sulfate (above) .
  • FIG. 16A, FIG. 16B, FIG. 16C and FIG. 16D show MRI 4.7 Tesla, Tl-weighted images of Copenhagen rats with the AT-1 prostate tumor model (as in FIG. 13A, FIG. 13B, FIG. 13C and FIG. 13D) , but rats are injected i.v. with Ferrioxamine Selective Contrast Agent, wherein the Active is non-covalently bound to a non-anticoagulant GAG, Heparan Sulfate, the Agent lyophilized and reconstituted with water just prior to administration at a concentration of 0.332 mmol/mL Fe(III) and administered at the usual dose of 0.155 mmol of Fe(III) per Kg.
  • FIG. 16A Precontrast.
  • FIG. 16D 40 MPI. Note the very homogeneous enhancement of Outer Rim and Central Tumor at virtually all post-contrast times, in relation to the differential Rim enhancement achieved by essentially all of the other GAG carriers. This property may be useful in certain diagnostic and/or therapeutic applications.
  • FIG. 17A is a control infrared (IR) spectrum of gadolinium diethylenetriaminepenaacetate (Gd:DTPA) (see Example 21) .
  • FIG. 17B is a control IR spectrum of N-methyl-1, 3- propanediamine (MPD) (see Example 21) .
  • FIG. 17C is a control IR spectrum of a mixed (and dried) solution of the individual chemical components, Gd:DTPA and MPD (1:1 molar ratio) .
  • FIG. 17D is the experimental IR spectrum of MPD covalently conjugated at a 1:1 molar ratio to DTPA (as described in Example 21) . Note the change in the height and splitting of the signature peak at 1400 wavenumber, and the change in the height and configuration of the broader stretching bands at 3300-3600 wavenumbers, which are indicative of covalent conjugate formation.
  • This T2 image is performed in order to identify the approximate locations of 2 tumor nodules (right posterior liver) and 1 tumor infiltrate (central liver region) , all tumor growths being confirmed at necropsy by gross visual inspection.
  • FIG. 18B Tl Precontrast image of liver (tumor not conspicuous) .
  • FIG. 18C Tl liver image a 7 MPI, Gd:MPD- DTPA:dermatan sulfate selective contrast agent (0.155 mmol/Kg) , showing extremely strong contrast enhancement of 2 solid tumor nodules (right posterior liver) and 1 irregular tumor infiltrate (central liver region) , in the identical locations as those indicated by the T2-weighted scout image (FIG. 18A) , but with much better definition of the tumor margins and much higher contrast gradients at the tumor margins.
  • FIG. 18D and FIG. 18E Tl Liver image at 20 and 40 MPI, Gd:MPD-DTPA:dermatan sulfate selective contrast agent (0.155 mmol/Kg) , showing continued very marked contrast enhancement of the 2 solid tumor nodules (right posterior liver) and the 1 irregular tumor infiltrate (central liver region) , with continued very highly demarcate ⁇ tumor margins and essentially no contrast fading.
  • FIG. 18F Tl Liver image at 20 and 40 MPI, showing continued very marked contrast enhancement of the 2 solid tumor nodules (right posterior liver) and 1 irregular tumor infiltrate (central liver region) , with only a very slight degradation in the sharpness of tumor margins over 40 MPI, only a very minimal decrease (fading) of tumor contrast intensity in the 2 solid nodules (right posterior liver) , a further brightening of the tumor infiltrate (central liver region) , and a very slight background brightening of surrounding uninvolved liver.
  • FIG. 19A Precontrast image for Gd:MPD- DTPA:dermatan sulfate selective contrast agent, showing only the tumor and superficial back fat and back muscle, because a surface coil is used and not a whole body coil.
  • FIG. 19B Post-contrast image, 7 MPI i.v. of Gd:MPD-DTPA:dermatan sulfate selective contrast agent, liquid form. Note the extremely strong enhancement of the entire tumor mass and the extremely strong gradient at the boundary between tumor and underlying normal tissue (image right) .
  • FIG. 19C Post-contrast image, 20 MPI i.v. of
  • Gd:MPD-DTPA dermatan sulfate selective contrast agent, liquid form. Note the extremely strong enhancement of the entire tumor mass and the extremely strong contrast gradient at the boundary between tumor and underlying normal tissue. Contrast has decreased slightly in the central tumor region, such that the tumor neovascular array is now very well visualized.
  • FIG. 19D and FIG. 19E Post-contrast image, 40 and 60 MPI, of Gd:MPD-DTPA:dermatan sulfate selective contrast agent, liquid form. Note the still very strong enhancement of the tumor, and particularly the retention of an extremely strong contrast gradient at the boundary between tumor and underlying tissue.
  • the basal rim (image right) is relatively protected from this T2* darkening artifact, due to more rapid backdiffusion of the agent into plasma at this basal site. Hence, moderately lower doses are indicated.
  • FIG. 20 shows a special histochemical stain
  • the low 60-minute staining of extracellular matrix may result from either or both of: (a) a more diffuse distribution of metal ions at 60 minutes (versus 7 minutes in FIG. 6 and FIG. 7A) , diffuse metal ions being more difficult to visualize (due to their smaller optical staining niduses) ; or (b) plasma backdiffusion of a portion of the initially localized metal.
  • FIG. 21A Frozen 8-micron thick section of prostate adenocarcinoma (outer 2/3 of tumor of ca. 4 cm diameter) , excised from its host rat (Copenhagen strain, syngeneic) 3 hours after intravenous injection of 5 mg/Kg doxorubicin:DS (doxorubicin in association with essentially purified dermatan sulfate, 435 Type, Opocrin) at a weight ratio of 60:40 (doxorubicin to dermatan sulfate) , and fluorescence microscopy performed using a rhodamine-type filter to elicit direct fluorescence of the doxorubicin drug substance (see also Example 29) .
  • DS doxorubicin in association with essentially purified dermatan sulfate, 435 Type, Opocrin
  • 60:40 doxorubicin to dermatan sulfate
  • fluorescence microscopy performed using a rhod
  • FIG. 21B Section of same tumor as in FIG. 21A, but in a subregion with looser clusters of tumor cells which are located and arranged around an endothelial stalk (oriented horizontally across the image) . Note the very bright staining of almost all cells, plus the strikingly bright fluorescence of doxorubicin now localized at nuclear sites, as well as in the tumor-cell cytoplasm. Also note the strong fluorescence of endothelial cells and endothelial-cell nuclei.
  • FIG. 21C Section of the same tumor type and size, but from a different Copenhagen rat, which was sacrificed 3 hours after intravenous injection of 5 mg/Kg of standard doxorubicin (Adriamycin PFS liquid) .
  • the present invention involves nontoxic, biodegradable small molecules, particles or microspheres (less than about 0.2-100 micrometers (um) in size) and microaggregates (1-200 nanometers, nm) comprising endothelial-binding substances and in particular, dermatan sulfate.
  • compositions of matter serving as formulation carriers for efficient, nonmagnetic drug localization in normal and diseased tissues, including microspheres or nanospheres comprising a special class of dermatan sulfate as described herein which binds to the complementary heparins and heparan sulfates present on normal endothelium throughout the body.
  • This invention is not considered to be constrained by prior art involving the formulation of microcarrier matrices from any of the presently proposed materials providing that the said materials were not previously recognized and documented in vivo as undergoing multiple endothelial binding and inducing rapid endothelial envelopment, and producing accelerated extravasation of macromolecules, microaggregates and microparticles in either the first microvascular bed encountered, or potentially (as proposed) semiselectively at foci of disease following systemic intravenous administration.
  • Endothelial-envelopment carriers may be formulated and stored in either the dry or fluid state, to which may be added, for example, pharmaceutically acceptable appropriate stabilizers, osmotic agents, colorings, flavorings and physiologic solutions which render them appropriate for intravascular and intracavitary injection.
  • the present invention is envisioned as most particularly applying to the vascular targeting phase of any future device (see below) which is developed for the efficient first-step transit across the external body barriers (e . g. , gastrointestinal tract; oral, nasal rectal, bladder or vaginal mucosa; skin, cornea or sclera) .
  • the present disclosure documents that drug carriers which comprise microencapsulation spheres with surface adhesion properties were selectively taken up into tissues by endothelial bioadhesion and by induced transendothelial migration, into the tissue interstitium.
  • the present application additionally documents that drugs controlled by such carriers, are deposited in selected target tissues, such as lung, in exact proportion to the deposition of drug carriers.
  • soluble drug-carrier complexes give comparable tissue uptake of drugs, under conditions in which the drug alone is not taken up.
  • the same and similar carriers are taken up by the transepithelial route in the lungs, gastrointestinal tract and bladder. Finally, it is established that the same and similar carriers undergo preferential lesional concentration in tumors and niduses of pulmonary infection.
  • novel carriers afford high-efficiency tissue uptake and localization of drugs, in particular, when the drugs are controlled by nonembolizing (less than 3-4 ⁇ m) carriers.
  • the carriers are preferably of a non-embolizing size of less than 500 nm and more preferably less than about 250 nm.
  • these carriers a) are formulated of water-soluble, biocompatible and biodegradable materials, and b) afford widespread percolation throughout tissue interstitium (and lesional gels) in a fashion which is not possible for hydrophobic carriers (e.g., liposomes) .
  • the carriers of the principal embodiments interact with their initial sites of cellular uptake (endothelial and epithelial cells) based on carbohydrate-carbohydrate binding and they do so in such a fashion as to produce multivalent binding, which leads to an induced, active endothelial (or epithelial) envelopment and transendothelial (or transepithelial) transport of both the carriers and drugs controlled by the carriers.
  • This preferably involves transcytosis (process occurring across one endothelial or epithelial cell) or may involve endothelial (epithelial) migrational overgrowth of the carriers, leading to envelopment .
  • multivalent binding to cells must occur, in order to induce active extravasation (or epithelial transport) of the drug- carrier couple, wherein such transport is significantly accelerated relative to that obtained for uncoated (uncontrolled) particles or drug-carrier complexes; this acceleration being of such a degree that transcellular transport of nonembolizing as well as embolizing particles (complexes) is completed within twelve minutes of endothelial/epithelial contact (typically in less than 5 minutes) , under in vivo conditions of microvascular blood flow and/or cavitary fluid flow, air flow, or enteric flow (in microvessels, bladder, lungs, bowel, or other body -cavities, respectively) .
  • the carriers must control the delivery : multipl (at least two) molecules of drug, in order to distmguir:. them from naturally transported simple hormones, proteins, peptides, and hybrid conjugates of two low- molecular-weight drugs.
  • TDMAC heparin tridodecyl methylammonium chloride heparin
  • GAG's glycosaminoglycans
  • the 8-12 unit fragment of dermatan sulfate binds heparin cofactor II without activating it. Unlike native heparin, neither dermatan sulfate nor its 8-12 unit fragment inhibits the constitutive endothelial surface coagulant, antithrombin III. This is also true of the shorter, semisynthetic fragments of heparin. Hence, dermatan sulfate and the short fragments of both heparin and dermatan sulfate, are envisioned as having even less anticoagulant activity than does native heparin (whose minimal anticoagulant activities are still acceptably low in this regard, when the heparin is incorporated into drug microspheres and complexes) .
  • doxorubicin is preferably provided as doxorubicin HCl in order to promote ion pair binding or complexation with the sodium salt form (preferably) of dermatan sulfate. Therefore, doxorubicin and doxorubicin HCl are used interchangeably when describing the active agent in the drug carrier compositions of the present disclosure. Selective high- efficiency uptake of this drug and carrier complex is documented in the present application following administration of the complexed agent.
  • dermatan sulfate-doxorubicin The absence of endothelial injury by dermatan sulfate-doxorubicin is also documented. This novel result established the rationale for reformulating existing drugs Using dermatan sulfate and related kits (as devices) , which can be performed by hospital pharmacists on-site, just prior to drug administration. This new approach can allow localized tissue (lesional) uptake of drugs controlled by nonembolizing carriers, as follows: a) by intravenous administration to the lungs (high efficiency delivery) and systemic lesional sites (moderate efficiency delivery) ; or
  • the present application describes that secondary tissue percolation of these hydrophilic drug-carriers occurs in normal target tissues for dermatan sulfate- coated microspheres (interstitium, lymphatic and epithelial) .
  • additional examples are presented, which establish the general principal that, unlike the situation for lipid microemulsions, liposomes and other hydrophobic carriers, the present hydrophilic spheres percolate extensively through the interstitium of a tumor and the lesional gel of a spontaneous pneumonitis, to reach both the outer spreading rims and the inner necrotic cores of these lesions .
  • This provides new rationale for improved lesional penetration, cellular (microbial) access and uptake of drug carriers, and their entrapped (controlled) drugs. It is envisioned as allowing improved drug access to tumor cells and microorganisms lying in sequestered sites.
  • the present invention describes new entrapments of substances such as:
  • the present invention includes formulations which employ additional detergents as excipients for preparing the internal drug nanoparticles, nanoemulsions, or other internally entrapped, controlled-release subcapsules, complexes or agents for formulation and entrapment of the internal drug emulsions.
  • additional detergents include:
  • the present invention teaches that cancers (and drugs) can potentially be treated (and localized) in an improved fashion by using the described technology.
  • the bioadhesion carriers set forth in the present application are envisioned as being preferred for the delivery of drugs which are highly toxic (certain antitumor drugs) ; drugs which are highly labile; agents which experience inappropriate biodistribution or poor tissue access due to their large molecular size or the presence of disseminated, competing receptors in the body; and anti- adhesion pharmaceuticals (as depot formulations, for the prevention of cancer-cell metastasis, prophylaxis of atherosclerosis, and inhibition of white-cell and platelet adhesion to vascular endothelium) .
  • the present invention includes the use of additional methods for matrix stabilizing and controlling the release of drugs.
  • thickening agents such as polylactic and polyglycolic acids, polyaminoacids, poly-L-lysine, polyethyleneimine, glycerol, polyglycerols or polyalchols (with or without heating or chemical reaction) , polyethylene oxides, biodegradable poloxamers or poloxamines (pluronics or tetronics) , poly-COOH compounds (polycarbols) , or polyamines.
  • microparticle formulation includes (particularly for the purposes of product scale-up) : preferably, extrusion of matrix (and/or surface) components through single (and/or coaxial) , sonified or air-stream-fractured micro-orifices (single or multiport) ; alternatively, aerosolization using hybrid, homogenization-spray drying apparatus.
  • the present invention includes additional methods of extracting the solvents used for phase emulsification and simultaneously crystallizing the matrices, surfaces and/or entrapment materials) : preferably, hexanes; alternatively, ethanol or methanol .
  • Additional methods of sterilization (and/or particle sizing) of the final (or subfinal) preparations include: preferably, for heat-stable agents: autoclaving at 120°C for 10-20 minutes; preferably, for heat-labile agents: submicron filtration of complexes and nanoparticles; and irradiation of particles larger than 0.22 um; alternatively, ultrasonification.
  • 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 si tu chelation of endogenous body metals.
  • the present invention specifically describes the preparation and utilization of novel contrast agents for magnetic resonance imaging.
  • novel contrast agents consist of paramagnetic metal chelates, as distinguished from metal-atom complexes, wherein the presently disclosed chelates are bound to glycosaminoglycans (GAG) .
  • GAG glycosaminoglycans
  • 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.
  • 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 Tl- 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 .
  • 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.
  • 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] .
  • ferrioxamine [Crumbliss, 1991]
  • basic or amine derivatives of the polyaminocarboxylate chelator diethylenetriaminepentaacetate (DTPA)
  • DTPA diethylenetriaminepentaacetate
  • DOTA 1,4,7,10- tetraazacyclododecane-N,N' ,
  • site localization may be so pronounced that the inherent potency ( in vi tro paramagnetic Rl) of the paramagnetic metal ion may not be crucial to obtaining optimal site-localized image contrast or spectral enhancement effects.
  • the present invention discloses pronounced Tl image contrast effects for the basic metal chelate, ferrioxamine, which by virtue of chelated Fe(III) ions, has a potency, or Rl relaxivity, of about 1.6-1.8 [mmol . sec] -1.
  • 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 vi tro Rl 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] , and as high as Rl ⁇ 7.5 [mmol.
  • Gd(III) is chelated to certain DTPA derivatives, including N-methyl-1, 3-propane diamine-DTPA as one preferred embodiment of a group of preferred DTPA- amine and DTPA-basic derivatives which can both (a) allow accelerated water diffusion and relaxation above that of DTPA; and (b) bind non covalently to acidic saccharides, including, preferably, glycosaminoglycans.
  • 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 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.
  • 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 e t al . '498, incorporated by reference herein] .
  • 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, modifications or fragments of the preceding substances, all defined herein as being subsumed under the names of the parent substances and categories.
  • 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, with the most preferred being the essentially purified dermatan sulfate as described herein.
  • the carriers of sulfated cyclodextrin, dextran sulfate and hyaluronic acid are included 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.
  • 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.
  • preferred basic chelators ferrioxamine and Gd(III) DTPA-lysine, and most preferred, N-methyl-1, 3- propane diamine-DTPA (N-MPD-DTPA) , are bound to their acidic glycosaminoglycan carriers at weight ratios of >, 70%.
  • Alternative covalent active-carrier conjugates may be preferred in certain instances, and preferred examples thereof are shown for MRI applications.
  • the preferred carrier substances from the standpoint of low toxicity and optimal safety margins at the higher doses which typify MRI contrast agent administrations are the dermatan sulfates with relatively low S0 3 -/COO- ratios of preferably between 0.7:1 and 1.8:1, most preferably between 0.9:1 and 1.5:1, and typically 1:1; and additionally with relatively low sulfur content of preferably less than 9% (w/w) , most preferably between 4% and 7% (w/w/), and typically 6.3- 6.4% (w/w); and the most preferred carrier substances under the high-dose administration conditions employed just above, comprise the new special class of dermatan sulfates with oversulfation of only selected oligosaccharide sequences but without overall oversulfation of the entire molecule (as described and defined above) .
  • Alternative preferred Agents 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, and also preferably comprising their alternative paramagnetic metal ion chelates, as disclosed above.
  • the carrier substance most preferably used in the present invention is the new class of essentially purified dermatan sulfates, enriched in uronic (L- iduronic) acid content and, in addition to its major monosulfated disaccharide sequence, (Ido-GalNAc4S0 3 ) , also characterized by an oligosaccharide sequence with a selectively high degree of sulfation, including the oversulfated saccharide sequences, (IdoA2S0 3 -GalNAc4S0 3 ) and (IdoAGalNAc4, 6S0 3 ) (as assessed by disaccharide analysis and as uniquely correlated with 1 H and 13 C magnetic resonance spectra) , enriched in heparin cofactor II activity, preferably greater than 220 Units/milligram, but low in factor Xa and antithrombin III activity and in overall anticoagulant activity (preferably less than 10% and most preferably less than 5% of standard heparin by USP
  • the dermatan sulfates of the preceding paragraph may, in one case, be prepared by the methods of: (a) grinding and treating animal organs or tissues, including beef mucosa, swine skin or lung, and preferably for certain of the present uses, beef mucosa, with proteolytic enzymes including papain, at pH 5 to 7 for the shortest possible time to remove proteins; (b) passage over a strong anion (basic) exchange resin including a acroreticular styrene-divinylbenzene matrix functionalized with quaternary ammonium groups and having a particle size range of 0.3 to 1.3 mm; (c) eluting the sulfated polysaccharides with a neutral salt solution between of 0.7 and 2.0 molarity; (d) crystallization of the dermatan sulfate as a low-solubility salt of a bivalent or trivalent metal including copper, iron and calcium, and preferably copper; (e) reconversion to sodium salt via c
  • dermatan species which is not intended in any way to limit the scope of the present invention, comprises a subspecies of these dermatan sulfates (sulphates) , as described [Mascellani, et al . WO 93/05074 (1993) , incorporated herein by reference; Mascellani, et al . (1994) , incorporated herein by reference] .
  • dermatan sulfate is the Type 435 beef mucosal dermatan sulfate (sulphate) manufactured and supplied by Opocrin S.P.A., Via Pacinotti, 3, 1-41040 Corlo Di Rormigine, Italy.
  • induced platelet aggregation, anticoagulation and bleeding which are characteristically induced by the more highly sulfated and/or longer-chain (higher molecular weight) carriers, including sulfated, oversulfated and polysulfated glycosaminoglycans and natural and synthetic sulfated, oversulfated and polysulfated polysaccharides and sulfatoids -- most specifically those with a sulfur content of 10% or greater, and those with a USP heparin- type anticoagulant activity ranging from 15 to 145 USP units per milligram or greater.
  • the preferred dermatan sulfates (above) and the most preferred new special dermatan sulfate subspecies, essentially purified as prepared by the special processes described above, when used as site-selective diagnostic or drug carrier substances, are clearly distinguished from all of the previous, older dermatan sulfates, i.e., those (a) not having the special structures described above; (b) not prepared according to the above isolation and purification processes; or (c) not prepared by such alternative processes as would give comparable enrichment of the preferred oligosaccharide sequences and selective sulfations described above.
  • dermatan sulfates are also clearly distinguished, when used as above, from all of the prior older dermatan sulfates in that they are not only structurally different, but they are also essentially free of the contaminating heparins, heparan sulfates and heparinoids which bind normal endothelium, undergo various degrees of in vivo metabolism, and interfere with rapid and complete blood and body clearance [Dawes, et al . (1989) , incorporated herein by reference] .
  • the new special dermatan sulfates described above are, when used as site-selective diagnostic or drug carrier substances, even more distantly distinguished from the non-dermatan sulfate classes of glycosaminoglycans, namely, (a) chondroitin sulfates A and C -- which do not share the uronic (L-iduronic) acid sugars of dermatan sulfate [Walton, et al . , US Patent 4,489,065; Maeda, et al .
  • hyaluronic acid which is a non-sulfated glycosaminoglycan
  • all of the polysulfated glycosaminoglycans and oversulfated sulfatoids e.g., bacterial polysulfates including pentosan polysulfate -- all of which characteristically have sulfur contents of 10% or greater that create significant in vivo safety issues due to polysulfate- induced platelet aggregation and cell membrane perturbation/lysis, or act as cofactors for such cellular lysis and which can affect normal body cells as well as tumor cells and other pathological cells/organisms, such as that specifically described as direct toxic cofactor "opening" of tumor cells produced by chondroitin polysulfate, resulting from chondroitin polysulfate- induced membrane damage [Landsberger (1984)] .
  • the new special dermatans preferred in the present invention are ones which do not themselves cause significant direct cellular or membrane damage, but instead induce rapid (3- to 7-minute) selective binding of disease-site endothelium, rapid (10 to 5-minute) endothelial cell transport, tumor uptake, deep matrix permeation and tumor-cell internalization of the attached diagnostic or drug active without the dermatan sulfate carrier itself or alone, damaging either the intermediate (e.g., endothelial) or final (e.g., tumor) target cells.
  • intermediate e.g., endothelial
  • final e.g., tumor
  • This new special class of dermatan sulfate is clearly distinguished from chondroitin sulfate Types A and C by its high content of L-iduronic (uronic) acid relative to the low or absent content in chondroitin sulfates A and C; and by its relatively lower modal molecular weight, most typically less than 25,000 daltons versus the chondroitin sulfates A and C, which typically equal or exceed 25,000 daltons modal molecular weight.
  • the relatively lower molecular weight of the new special dermatan sulfates has at least three surprising and unexpected advantages when used as a carrier substance for bound or associated active substances: (a) very rapid blood clearance of the carrier and active, predominantly by the renal route, with a blood t 1/2 of typically about 20 to 120 minutes, increasing only very gradually as a function of increasing dose; (b) minimal to absent in vivo metabolism -- in major contrast to standard heparins, heparan sulfates and chondroitin sulfates A and C -- thereby giving extremely low residual in vivo deposition or retention of the carrier material; and (c) maximal, rapid vascular egress across disease-site endothelium -- including across induced and "permeabilized" endothelium, e.g., induced by Vascular Endothelial Growth Factor/Vascular Permeability Factor (VEGF/VPF) for maximal disease-site and tumor access, uptake and tumor-cell internalization of the bound or associated active substance.
  • this new class of dermatan sulfates has been recognized as useful for conferring antithrombosis in the absence of (heparin-type) anticoagulant activity and bleeding side effects, it has not previously been recognized, nor would it be obvious to one skilled in the art, that this new special class of dermatan sulfates could confer the surprising and unexpected advantages of acting as a highly potent and effective in vivo carrier of noncovalently or covalently bound amine or chemically basic chelators or metal chelates, furthermore, to selectively localize them in sites of disease, including tumors, across non-permeabilized as well as "permeabilized” vascular endothelium and simultaneously to promote very rapid clearance of the non-targeted fraction of carrier plus active, highly preferentially by the renal route, in a fashion which increases only very gradually with increasing dose -- thereby conferring not only reduced side effects and low in vivo retention, but also the additional advantages of: (a) very low imaging backgrounds at very early times post-injection upon intravenous administration for the purpose
  • the new special dermatan sulfates which can preferable allow a noncovalent method of binding the active to the carrier, and hence, can enable a high quantity of active to be bound per unit of carrier, preferably greater than 70% (weight % of active to [active + carrier] ) versus typically 7 to 12% (w/w) for most covalently bound active-polymer systems, including antibody systems.
  • the self-assembling, noncovalent formulation (as well as covalent formulation) properties of the new special dermatan sulfates provide an additional surprising and unexpected advantage of minimizing the quantity of dermatan sulfate carrier required to administer and selectively localize an effective in vivo dose of paramagnetic chelate.
  • 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.
  • essentially purified dermatan sulfate (435 Type of 17,000 to 19,000 modal MW, with selectively oversulfated oligosaccharides and a heparin cofactor II activity at least about 220 U/mg, Opocrin) , is used in noncovalent (or covalent) association with the following oncology actives to localize them in sites of disease and facilitate their clearance them from the rest of the body: doxorubicin, adriamycin, taxol, vincristine, vinblastine, bleomycin, idarubicin, epirubicin, amsacrine, azacitidine, dideoxyinosine, dihydro-5- azacytidine, ethanidazole, ethiofos, methotrexate, misonidazole, porfiromycin, pyrazoloacridinek, terephthalamidine, taxotere and other taxane derivatives, to
  • essentially purified dermatan sulfate (435 Type of 17,000 to 19,000 modal MW, with selectively oversulfated oligosaccharides and a heparin cofactor II activity at least about 220 U/mg, Opocrin) , is used in noncovalent (or covalent) association with the following anti- infectives: gentamicin, amikacin, tobramycin, and other amine, basic, basic peptidic, basic polypeptidic, hydrophobic or amphoteric antibiotics or bacterial, fungal, mycobacterial, viral or other microbial or microbiological diseases.
  • compositions substantially similar to those disclosed above may be carried out using compositions substantially similar to those disclosed above.
  • alternative metal ions may be chelated for purposes of metal-ion exchange at the site.
  • the present formulations may contain or comprise metal ions of manganese, aluminum, germanium, zinc, cobalt, calcium, platinum, or others.
  • such compositions may contain or comprise metal ions of boron, cobalt, rubidium, yttrium, technetium, ruthenium, rhenium, indium, iridium, thallium, samarium or others.
  • 59 Fe and 67 Ga 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.
  • 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.
  • deferoxamine is precipitated out of aqueous salt solutions by the addition of 2 N KHC0 3 , as previously reported [Ramirez et al . (1973) , incorporated by reference herein] .
  • a saturated solution of deferoxamine (320 mg/mL at 25°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°C in an ice bath and 2.5 L of 2.0 N KHC0 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.
  • 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(0)OH (13.44% w/v of Fe(0)0H particles, Arthur Technologies Corporation, San Antonio, Texas), 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° C for between 1 and 24 hours and the pH adjusted periodically to between 6.5 and 7.9 by addition of 0.10 N NaOH. Formation of 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 (-2500 g) for 15 minutes to remove any unreacted or aggregated Fe(0)OH.
  • This final batch volume is adjusted as desired, typically to a final volume of 2600 mL. Any remaining trace amounts of unreacted Fe(0)OH are removed and the solution also made aseptic, by passing the supernatant through a 0.22 ⁇ m Millipore GV-type filter in a Class 100 laminar flow hood. The resulting batch is stored at 4°C in an autoclaved, sealed glass container until further use (see Examples below) .
  • DTPA 500 mg
  • L-Lysine hydrochloride powder 931 mg
  • 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 0.1 N HCl.
  • the water- soluble carbodiimide, l-ethyl-3- (3- dimethylaminopropyl) carbodiimide HCl (EDO , 732.5 g, is dissolved in 2 mL water and its pH also adjuste as above.
  • This EDC solution is added dropwise to the DTPA - lysine solution mixture (above) over I hc r at Z 2 ° C witr. constant stirring and periodic adjustment of pH to 4.7:, and the reaction allowed to proceed to completion over I more hours.
  • N-epsilon-t-BOC-L-lysine 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°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 x 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.
  • the gadolinium(III) chelate of DTPA-Lys namely Gd(III) :DTPA-Lys
  • 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 0.1-1.0 M, as needed, until a stoichiometric quantity of Gd(III) has been added.
  • the pH is adjusted to 7.0 by addition of 1.0 N NaOH.
  • gadolinium oxide can be added and the reaction mixture stirred for 24 hours. In the case of gadolinium oxide, neutralization with 1.0 N NaOH is not needed.
  • 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) .
  • 0.1 to 1.0 N 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 ⁇ 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°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°C for 15 minutes. Alternatively, further processing is carried out on the liquid by filling into glass vials, freezing at -50°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.
  • 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.
  • 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 ⁇ m filter to remove any residual insoluble iron oxides- hydroxides and render the liquid agent aseptic.
  • the aseptic agent is stored at 4°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.
  • 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
  • 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 0.10 N HCl. l-ethyl-3- (3- dimethylaminopropyl)carbodiimide HCl (EDO, 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 0.10 M HCl. Aliquots of the EDC solution are added to the mixture of glycine-glycosaminoglycan over a one hour period.
  • 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 f ctor of [ (mEq sulfates/mg of carrier as above) / (mEq sulfates/mg of beef lung heparin*)] .
  • a scaling f ctor of [ (mEq sulfates/mg of carrier as above) / (mEq sulfates/mg of beef lung heparin*)] .
  • 0.1 to 1.0 N 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 ⁇ m filter to remove any residual insoluble iron oxides- hydroxides and render the liquid agent aseptic.
  • the aseptic agent is stored at 4°C.
  • 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.
  • 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.
  • additional acidic saccharides including sucrose octasulfate and sulfated cyclodextrins
  • glycosaminoglycans including keratan sulfate and hyaluronate
  • additional sulfatoids including the bacterial sulfatoid, dextran sulfate.
  • 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.
  • 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.
  • 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) .
  • SOS sucrose octasulfate
  • 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.
  • T ir results ir. formation of a stable covalent conjugate, tyrically via an amide bond between the active and t ⁇ e carrier.
  • Examples 12, 13, and 14 (below) describe the synthesis rt ferrioxamine-heparin covalent conjugates, wherein the ferrioxamine is covalently bound to heparin via three different coupling reagents.
  • Aqueous ferrioxamine 2.0 g, as prepared in Example 1, is adjusted to pH 4.75 by addition of 0.10 M HCl.
  • Beef-lung heparin Hepar-Kabi-Pharmacia, Franklin, OH
  • 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 re-adjusted to 4.75 with 0.10 M HCl.
  • the water-soluble carbodiimide, 1- ethyl-3- (3-dimethylaminopropyl) carbodiimide HCl (EDO , 2 g, is weighed into a scintillation vial, solubilized in a minimum amount of water, and the pH adjusted to 4.75 with 0.10 M HCl. Aliquots of EDC solution are pipetted into the mixture of ferrioxamine-heparin over a one hour period. After each addition of EDC the 0.10 M 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 ( ⁇ 2500 x 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 re-precipitating 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.
  • the activated EEDQ-activated heparin is collected by centrifugation at 4500 rpm ( ⁇ - 2500 x 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 activated intermediate of beef-lung heparin (Hepar-Kabi- Pharmacia, Franklin, OH) 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. Carbonyl- diimidazole (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°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.
  • CDI carbonyl- diimidazole
  • 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°C.
  • the deferoxamine-heparin conjugate is collected by centrifugation at 4500 rpm ( «- 2500 x 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 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.
  • 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.
  • cDTPAA diethylenetriaminepentaacetic dianhydride
  • Beef-lung heparin Hepar-Kabi-Pharmacia, Franklin, OH
  • cDTPAA 4.5 g ( ⁇ - 100 mole excess relative to heparin) , is weighed out and divided into 20 equal (225 mg) aliquots.
  • 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 0.10 M 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°C and stored at 4°C.
  • 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 2 0 3 or Fe(0)OH, and stirring for 24 to 36 hours at 37°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 ( « « 2500 g) , and the product separated from unreacted metal oxides by filtration through a Millipore 0.22 ⁇ m GV-type filter, followed by dialysis against 150 volumes.
  • concentrations of chelated metal ion and heparin are determined by inductively coupled plasma (ICP) and size- exclusion HPLC, respectively.
  • ICP inductively coupled plasma
  • HPLC size- exclusion HPLC
  • 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.
  • 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) .
  • Tl-weighted MRI images are performed at 1.0 and 1.5 Tesla, before (Pre) and after (Post) intravenous (i.v.) injection of Ferrioxamine :Dermatan Sulfate, 435 type 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 Tl-weighted Precontrast images.
  • the tumors (a) become rapidly and markedly enhanced at an early post-injection time (7 mins) (FIG. 2A, FIG. 2B) ;
  • FIG. 2B (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) (FIG. 4A, FIG. 4B, FIG. 4C, FIG. 4D, MRI images; FIG. 5, quantitative region-of-interest, ROI, analysis) with continued very well defined tumor borders at prolonged imaging intervals. Correlation of these MRI images with 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 (FIG. 6 and FIG. 7A) and not when administered in free form (Active alone) (FIG. 3A, FIG.
  • FIG. 8A, FIG. 8B and FIG. 8C 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 FIG. 8A, FIG. 8B and FIG. 8C are much longer than those typically reported for Gd:DTPA dimeglumine contrast enhancement at body organ sites.
  • Tl-weighted MRI images (TR/TE - 250/8) performed at 4.7 Tesla, before (Pre) and after (Post) intravenous (i.v.) injection of Ferrioxamine:Dermatan Sulfate, 435 type 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 (FIG. 9A, FIG. 9B, FIG. 9C, FIG. 9D, FIG.
  • 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.
  • 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) .
  • enhancement reverts to essentially background levels, and at 60 MPI, there is no residual contrast, except for central cystic regions.
  • Tl-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 435 type 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.
  • 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.
  • 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.
  • the diethylenetriamine-pentaacetic acid anhydride (DTPA anhydride) solution is prepared by adding 180 ml of anhydrous dimethylformamide (DMF) into a 250 ml round bottom flask.
  • the flask is fitted with a side arm addition funnel and contains a magnetic stir. While the DMF is stirring vigorously, 5 g (14 mmol) of DTPA anhydride (Sigma Chemical Co.) is added in 0.5 g portions over one hour.
  • the resulting suspension is warmed to 60°C to 15 minutes or until the solution clears.
  • the flask is removed from the heat and placed in an ice bath until the solution has equilibrated to 4°C.
  • the MPD-DTPA derivative is prepared by mixing 15 ml of DMF with 1.46 ml (14 mmol) of N-methyl-1,3 propanediamine (Sigma Chemical Co.) in the addition funnel.
  • the MPD-DMF mixture in the side arm addition funnel is added to the cold (4°C) , vigorously stirring DTPA anhydride solution, dropwise. A white precipitate forms throughout the addition.
  • the suspension is allowed to stir overnight at room temperature.
  • the MPD-DTPA derivative is collected by centrifugation at 2500g for 10 minutes and washed repeatedly with acetone (5 x 300 ml) .
  • the product at this stage, in concentrated solution has a pH of 3.5, additional purification requires a solution pH of 7.0.
  • the product MPD-DTPA derivative is dissolved in water and the pH is adjusted to 7 with 5 N NaOH.
  • the product is lyophilized for 16 hours to dryness.
  • the lyophilized material is dissolved in a minimum amount (40 ml) of warm (50°C) methanol for 15 minutes, cooled to room temperature, and precipitated with 10 volumes of acetone.
  • the precipitate is collected by centrifugation at 2500g for 10 minutes.
  • This material is again dissolved in warm methanol for 15 minutes, precipitated with 10 volumes of acetone and collected by centrifugation at 2500xg.
  • the precipitate is washed repeatedly with acetone, dried under nitrogen and stored in a vacuum desiccator.
  • N-Methyl-1,3- propanediamine-DTPA The chelating capacity of N-Methyl-1,3- propanediamine-DTPA (MPD-DTPA) is determined by titrating a small aliquot with 0.1 M GdCl 3 5H 2 0 in 1 M ammonium acetate (pH 5.5) buffer, using Xylenol Orange (5%, w/v) as the colorimetric indicator of endpoint . Based on this titration, a stoichiometric quantity of 1 M GdCl 3 5H 2 0 is added to a batch quantity of N-MPD-DTPA as follows: the bulk MPD-DTPA is dissolved in a minimum amount of water (ca. 300 mg/ml) , 1M GdCl 3 5H 2 0 is to the added while vigorously stirring, and the pH is adjusted from ⁇ 4.0 to - Ill -
  • the average chelating capacity is about 22% (by weight) , with slight variation based on the extremely hygroscopic nature of the dry chelator.
  • Very strong paired-ion binding is indicated by 73% retention of Gd within the bag for the Gd:MPD-DTPA:dermatan sulfate formulation prepared at 60:40% (Gd:MPD-DTPA to dermatan sulfate) ; compared to the much lower 23% retention within the bag for Gd:DTPA:dermatan sulfate when prepared at the same molar ratio of Gd:DTPA to dermatan sulfate.
  • the Rl potencies (Tl relaxivities) of (a) Gd:MPD- DTPA alone and (b) the 60:40% (w/w) paired-ion formulation of Gd: PD-DTPA:dermatan sulfate, are evaluated using an IBM PC20 Minispectrometer, and both are determined to be 7.8 mmol ⁇ s "1 (based on parallel determinations of Gd concentration by ICP atomic absorption).
  • the equality of Rl's for the Gd chelate alone and Gd chelate bound to dermatan sulfate, indicate that binding of the chelate to dermatan sulfate does not interfere with water diffusion and paramagnetic relaxation.
  • the average LD50 11.0 mmol/kg (of Gd and chelator) , with 3 mice surviving at an average of 9.9 mmol/kg and 3 mice dying at an average of 12.2 mmol/kg.
  • the LD50's were moderately lower in dose.
  • the formulations of Examples 2, 5, 21 and 22 are modified such as to bind the radioactive single-photon- emitting • (SPECT) metals, 67Ga or lllln, in place of the non-radioactive metal ions, Fe(III) or Gd(III) .
  • SPECT radioactive single-photon- emitting •
  • the dose of DFo:dermatan sulfate is increased 100X from 1.55 umole/kg to 155 umol/kg (0.155 mmol/kg) while maintaining the dose of radionuclide constant at 200 uCi per rat, in order to assess the effects of MRI doses, dose augmentation and potentially therapeutic doses, on clearance half times.
  • clearance is very nearly identical to the 100-fold lower dose of agent (above) , with only a very minimal, ca. 5-minute prolongation.
  • lllln is converted to the acetate form at pH 5.5-6.5, used to radiolabel MPD-DTPA:dermatan sulfate (60:40 wt % MPD-DTPA:dermatan sulfate, 435 Type, Opocrin) .
  • Clearance times and organ clearance patterns (renal versus liver) are comparable to those of 67GaDFo:dermatan sulfate (above) ; and when tested, tumor uptake is also rapid and distinct.
  • Gadolinium N-methyl-l,3,propanediamine-
  • a T2 scout image (TR/
  • FIG. 18C display rapid and prolonged (through 60 minutes) sharp tumor boundaries against the surrounding uninvolved liver (FIG. 18C, FIG. 18D, FIG. 18E) , and exhibit prolonged (sustained) contrast through 60 minutes (FIG. 18F) , with only a very slight degradation of the contrast gradient at the tumor boundaries at 60 minutes postinjection (MPI) .
  • MPI postinjection
  • the MRI contrast enhancement produced by Gd:MPD-DTPA:DS is markedly greater (more potent on a dose basis) than that produced by the ferrioxamine:dermatan sulfate agent of Example 18; and is slightly to moderately greater (more potent on a dose basis) than that produced by Gd:DTPA-lysine:dermatan sulfate (prepared per Examples 3, 4 and 9; see also FIG. 13A, FIG. 13B, FIG. 13C, FIG. 13D, Example 21 and Table 2 for relative potency) ; both of the preceding agents containing less potent metal chelates, namely, with Rl's of 1.6 and 4.2, respectively, compared to an Rl of 7.8 [mmol.
  • the images of the present Example show all the following, surprising and unexpected advantages over Gd:DTPA (dimeglumine) , as well as over all the reported liver-specific Tl and T2 contrast agents: (a) uptake by tumor proper without substantial uptake by the surrounding uninvolved liver; (b) enhanced tumor selectivity and sensitivity; (c) prolonged as well as immediate tumor uptake, for improved clinical flexibility of multi-site and multi-image acquisition without contrast fading or need for multiple contrast-agent injections; (d) improved contrast sharpness and brightness gradient at the tumor boundaries, for improved tumor staging and improved detection of small tumors; (e) improved detection of small metastases; and (f) improved detection of small invasive outgrowths, for enhanced prognostic and therapeutic monitoring information.
  • Gadolinium N-methyll-1,3,propanediamine-
  • Gd:MPD-DTPA:DS produces a rapid, extremely strong Tl contrast enhancement of the entire tumor at 7 minutes (FIG.
  • TR/TE 2500/250
  • Example 18 The surprising and unexpected advantage of endothelial localization observed here for malignant prostate tumor, was also observed in Example 18 for malignant breast tumor. This corroborates the surprising and unexpected finding of Example 18 above, that tumor-induced neovascular endothelium, as well as tumor cells proper, are targets for binding, pumping, extravasation and tumor-cell internalization of the dermatan sulfate-bound (including non-covalently bound) classes of MRI contrast agents, and indeed for other active agents similarly bound to dermatan sulfates and GAGs.
  • These findings of tumor endothelium, tumor matrix, tumor cell and nuclear localizations and accumulations further provide the basis for selectively localizing therapeutic agents, whether metal chelates or other types of active substances.
  • a 0.22 um low binding filter Millipore, Millex GV
  • the lyophilized cakes are stable (in both doxorubicin and dermatan components) for long intervals at room temperature as well as 4°C (doxorubicin analysis by HPLC: C-18 Lichrosphere; mobile phase: acetonitrile:water (50:50) , 20mM phosphoric acid + 5mM sodium dodecylsulfate, pH 2.3; dermatan sulfate analysis by
  • HPLC TSK molecular sieve
  • mobile phase 0.2M sodium sulfate for charge suppression, with Opocrin dermatan molecular weight standards of 1,800-17,250 daltons
  • the reconstituted cakes meet USP specifications for oversized particles above 10 and 25 um (by Hyac-Royco laser analysis) .
  • Adriamycin (Adria Laboratories source of doxorubicin) is also similarly prepared in paired-ion couples with dermatan sulfate (Opocrin, as above) .
  • Hepar-Kabi-Pharmacia Beef lung heparin (Hepar-Kabi-Pharmacia) is solubilized, mixed with high-purity doxorubicin, over the ranges of 90:10 to 10:90 ratios (w/w, doxorubicin:heparin) and at one of the optimal ratios, namely 60:40 (w/w) , and then subjected to the additional steps as described in EXAMPLE 28.
  • a visually clear, 0.22 um filterable liquid results. However, this liquid has a larger limit size of 25 nanometers by laser light scattering; and the lyophilized cake is considerably more resistant to rapidly homogeneous reconstitution (requiring ca. 1-2 hours) .
  • formulations are prepared in two steps, first by solubilizing and preparing the taxol in lecithin or lecithin-cholesterol nanodispersions, and second, by interacting the lecithin-coated nanodispersions with dermatan sulfate (435 Type, Opocrin) or beef lung heparin (Hepar-Kabi-Pharmacia) to produce and stabilize the final nanodispersions by paired-ion interaction at the nanoparticle surface, with binding of the glycosaminoglycan sulfate groups to the highly basic nitrogen groups of lecithin.
  • dermatan sulfate 435 Type, Opocrin
  • beef lung heparin Hepar-Kabi-Pharmacia
  • Dermatan sulfate or alternatively beef lung heparin
  • the mixture probe sonicated for 1 minute at 4°C is added over a range of 2-6% (w/w, to lecithin) and the mixture probe sonicated for 1 minute at 4°C.
  • Azure A the cationic dye
  • Vincristine (Sigma Chemical Cz . , St. :-:r' is dissolved in water and mixed with dermatan sulfate at ratios of between 90:10 and 30:70 (w/w) drug to dermatar. sulfate. This results in clear solutions, with an optimal ratio occurring at 30-40% (w/w) of drug, with particles being undetectable (by laser light scattering) .
  • This result in combination with retention of the paired- ion form, but not the drug alone, inside a 500 MW cutoff dialysis bag, is indicative of a strong paired-ion formation between the amine group of vincristine and the sulfate groups of dermatan sulfate.
  • Amikacin, gentamicin and tobramycin are dissolved in water and mixed with either dermatan sulfate (435 Type) or beef lung heparin (Hepar-Kabi-Pharmacia) at ratios of between 90:10 and 30:70 (w/w) drug to glycosaminoglycan.
  • the basic, white-cell chemoattractant, and inflammatory peptides (a) N-formyl-met-leu-phe-lys (acetate) , (b) arginine bradykinin (arg-pro-pro-gly-phe- ser-pro-phe-arg) and (c) poly-L-lysine (all 3 from Sigma Chemical Co., St. Louis), are dissolved in water and mixed with essentially purified dermatan sulfate (435
  • Type, Opocrin at ratios of 90:10 to 10:90 (w/w of active substance to dermatan sulfate) .
  • the paired-ion doxorubicin:dermatan sulfate (essentially purified dermatan sulfate, 435 Type,
  • standard doxorubicin liquid Adria Laboratories
  • Doxorubicin DS 0.01-0.02 0.05-0.06
  • doxorubicin has an acute murine toxicity which is at least comparable to and trending towards superior over standard doxorubicin, although the differences in this present test are not statistically significant.
  • doxorubicin:dermatan sulfate essentially purified dermatan sulfate, 435 Type, Opocrin
  • doxorubicin:DS standard doxorubicin liquid
  • Adria Laboratories doxorubicin liquid
  • the rats are sacrificed at 3 hours after injection, the tumors and major organs removed, the cut tumors & organ pieces placed in OCT polymer and frozen at 4°C, and cryostat sections cut at 8 um and coverslipped.
  • Fluorescence microscopy is performed by exciting the sections using a rhodamine-type bandpass filter (at ca. 485nm -- in order to selectively excite doxorubicin) and assessing direct doxorubicin fluorescence (at an emission wavelength greater than 530nm) to determine the following:
  • tumor targets i.e., endothelium cells as well as tumor cells proper
  • Doxorubicin essentially
  • FIG. 21B for doxorubicin:DS -- looser clusters of tumor cells on an endothelial stalk. Looser tumor-cell clusters are most likely to be in growth phase or division (compared to the more dense tumor-cell sheets of FIG. 21A) . Note the very bright staining of almost all cells, plus the strikingly bright nuclear fluorescence of doxorubicin now localized at this site, as well as in the cytoplasm. Also note the strong fluorescence of endothelial cells and endothelial-cell nuclei.

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Abstract

Compositions supports de médicaments consistant en un médicament complexé à un sulfate de dermatan. Le médicament est de préférence un antitumoral tel que le taxol, un peptide oncoagent ou la vincristine, ou mieux, la doxorubicine. Le sulfate de dermatan, pratiquement pur, contient jusqu'à 9 % en poids de soufre et présente une sursulfatation sélective par les oligosaccharides. Ces compositions qui s'administrent de manière à accéder facilement au système vasculaire y induisent in vivo les effets suivants: (1) enveloppement endothélial rapide, partiel ou total du support (diagnostique) du médicament; (2) emprisonnement du support, et protection de l'agent captif vis-à-vis de la clairance sanguine vasculaire anticipée (2 minutes) alors que la cavité endothéliale enveloppant le support s'invagine encore dans le compartiment vasculaire; (3) accélération du transport du support dans et/ou à travers les structures vasculaires endothéliales ou sous-endothéliales en direction du compartiment tissulaire (intersticium); et (4) amélioration de l'efficacité de migration du médicament à travers l'endothélium ou les barrières épi-endothéliales ou sous-endothéliales permettant de diminuer la dose nécessaire pour obtenir les effets désirée, par rapport à l'utilisation d'agents usuels. L'absorption par des tissus analogues et la migration trans-épithéliale dans les poumons, la vessie ou les intestins sont également décrits.
PCT/US1994/014776 1994-12-22 1994-12-22 Complexes de sulfate de dermatan et de medicaments, ameliorant la pharmacocinetique WO1996019242A1 (fr)

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WO2001093911A3 (fr) * 2000-06-08 2002-03-07 Trustess Of The University Of Complexes macromoleculaires de medicaments et compositions contenant ces complexes
WO2003074088A2 (fr) 2002-03-06 2003-09-12 Biotechnologie - Gesellschaft Mittelhessen Mbh Couplage de substances a faible poids moleculaire avec un polysaccharide modifie
AP1480A (en) * 2000-01-31 2005-10-31 Univ Of Arkansas A concentrated non-foaming solution of quaternary ammonium compounds and methods of use.
US7538092B2 (en) 2002-10-08 2009-05-26 Fresenius Kabi Deutschland Gmbh Pharmaceutically active oligosaccharide conjugates
US7541328B2 (en) 2002-03-06 2009-06-02 Fresenius Kabi Deutschland Gmbh Coupling proteins to a modified polysaccharide
WO2009039996A3 (fr) * 2007-09-11 2009-10-29 Mondobiotech Laboratories Ag Utilisation d'un peptide en tant qu'agent thérapeutique
US8840879B2 (en) 2004-03-11 2014-09-23 Fresenius Kabi Deutschland Gmbh Conjugates of hydroxyalkyl starch and a protein

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Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AP1480A (en) * 2000-01-31 2005-10-31 Univ Of Arkansas A concentrated non-foaming solution of quaternary ammonium compounds and methods of use.
WO2001093911A3 (fr) * 2000-06-08 2002-03-07 Trustess Of The University Of Complexes macromoleculaires de medicaments et compositions contenant ces complexes
US6417237B1 (en) 2000-06-08 2002-07-09 The Board Of Trustees Of The University Of Illinois Macromolecular drug complexes and compositions containing the same
WO2003074088A2 (fr) 2002-03-06 2003-09-12 Biotechnologie - Gesellschaft Mittelhessen Mbh Couplage de substances a faible poids moleculaire avec un polysaccharide modifie
WO2003074088A3 (fr) * 2002-03-06 2004-08-26 Biotechnologie Ges Mittelhesse Couplage de substances a faible poids moleculaire avec un polysaccharide modifie
US7541328B2 (en) 2002-03-06 2009-06-02 Fresenius Kabi Deutschland Gmbh Coupling proteins to a modified polysaccharide
EP2269655A3 (fr) * 2002-03-06 2011-04-27 Fresenius Kabi Deutschland GmbH Coulage de substances à bas poids moléculaire à l'hydroxyalkylamidon
US8916518B2 (en) 2002-03-06 2014-12-23 Fresenius Kabi Deutschland Gmbh Coupling proteins to a modified polysaccharide
US7538092B2 (en) 2002-10-08 2009-05-26 Fresenius Kabi Deutschland Gmbh Pharmaceutically active oligosaccharide conjugates
US8840879B2 (en) 2004-03-11 2014-09-23 Fresenius Kabi Deutschland Gmbh Conjugates of hydroxyalkyl starch and a protein
WO2009039996A3 (fr) * 2007-09-11 2009-10-29 Mondobiotech Laboratories Ag Utilisation d'un peptide en tant qu'agent thérapeutique

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EP0794796A1 (fr) 1997-09-17

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