WO1994016729A1 - Modulation ciblee de synthase d'oxyde nitrique ou de voie d'oxyde nitrique - Google Patents

Modulation ciblee de synthase d'oxyde nitrique ou de voie d'oxyde nitrique Download PDF

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Publication number
WO1994016729A1
WO1994016729A1 PCT/US1994/000894 US9400894W WO9416729A1 WO 1994016729 A1 WO1994016729 A1 WO 1994016729A1 US 9400894 W US9400894 W US 9400894W WO 9416729 A1 WO9416729 A1 WO 9416729A1
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nitric oxide
target cell
cell population
targeting
cells
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PCT/US1994/000894
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English (en)
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Donald B. Axworthy
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Neorx Corporation
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • 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
    • A61K47/62—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 a protein, peptide or polyamino acid
    • A61K47/66—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 a protein, peptide or polyamino acid the modifying agent being a pre-targeting system involving a peptide or protein for targeting specific cells
    • A61K47/665—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 a protein, peptide or polyamino acid the modifying agent being a pre-targeting system involving a peptide or protein for targeting specific cells the pre-targeting system, clearing therapy or rescue therapy involving biotin-(strept) avidin systems
    • 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
    • A61K47/68—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 antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—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 antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6849—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 antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention is directed to targeted modulation of a nitric oxide pathway or nitric oxide synthase to achieve therapeutic goals. More specifically, the present invention is directed to targeted control of nitric oxide producing and utilizing cellular mechanisms that are mediated by nitric oxide synthase or other catalysts as well as other nitric oxide synthase-mediated reactions.
  • nitric oxide has been identified as endothelium-derived relaxing factor (EDRF) which acts as a potent vasodilator by relaxing vascular smooth muscle through the activation of soluble guanylate cyclase and the cGMP cascade.
  • EDRF endothelium-derived relaxing factor
  • Brain and liver cells also employ active nitric oxide pathways.
  • nitric oxide is synthesized from L-arginine in a reaction mediated by the enzyme nitric oxide synthase.
  • Nitric oxide synthase mediates other cellular reactions such as the conversion of L-arginine to L-citrulline.
  • a plurality of nitric oxide synthase isozy es have thus far been identified.
  • Nitric oxide synthase may be induced by cytokines, such as tumor necrosis factor and interleukin-1, in vascular smooth muscle cells, for example. Extended duration nitric oxide production has been achieved in cells by degradation of S-nitroso-nucleophiles over time.
  • vascular reconstruction has been shown to be largely the result of proliferation of arterial smooth muscle cells in the intima in response to endothelial denudation.
  • Many possible contributors to the proliferative effect have been investigated, such as platelet aggregation, platelet-derived growth factor (PDGF) and the lack of EDRF. Loss of vascular response is also observed in septic shock and during tumor therapy using cytokines.
  • PDGF platelet-derived growth factor
  • the present invention is directed to agents capable of modulating a nitric oxide pathway of a target cell population to achieve a therapeutic effect.
  • agents include a targeting portion capable of delivering the agent to a target site and an effector portion capable of modulating a nitric oxide pathway at the target site.
  • Another aspect of the present invention includes agents capable of modulating nitric oxide synthase, thereby providing modulation of receptor-mediated reactions in which the enzyme plays a role.
  • additional agents useful in the practice of the present invention include free
  • agents of the present invention may be formed as conjugates or fusion proteins. Agents of the present invention may also be composed of two or more separately administrable moieties. In one embodiment, the first exhibits a targeting portion-ligand composition and the second has an anti-ligand-effector moiety structure. In another embodiment, nitric oxide synthase-targeting portion is administered, followed by effector moiety administration.
  • Preferred targeting portions of the present invention include monoclonal antibodies or other targeting proteins directed to cells with constitutive or inducible nitric oxide pathways.
  • Exemplary of such preferred targeting portions include monoclonal antibodies or other targeting proteins directed to vascular smooth muscle cells, corpora cavernosa smooth muscle cells, endothelial cells, brain cells, and liver cells as well as monoclonal antibodies or other targeting proteins directed to tumor cells.
  • Preferred effector portions of agents of the present invention include moieties capable of modulating a nitric oxide pathway.
  • Preferred effector portions include nitric oxide synthase or moieties capable of mediating the action of nitric oxide synthase enzyme(s) (e.g.. L-arginine modified with imidazole, acetylene or allyl functional groups) .
  • Exemplary preferred effector portions of agents of the present invention include nitrate compounds, L-arginine, poly-L-arginine, N G -hydroxy-L-arginine or polymers thereof, N G -hydroxy-N G -methyl-L-arginine, other arginine analogs, glutamic acid, poly-glutamic acid, N-methyl-D-aspartate, poly-N-methyl-D-aspartate, glucocorticosteroids, heme, non- he e iron or compounds which facilitate site-specific delivery of molecular oxygen or superoxide anion.
  • Other preferred agents of the present invention include antibodies or other targeting proteins that have undergone sulfhydryl modification to form S-nitroso moieties or, for example, L-arginine nitrogen modification to form nonoate equivalents.
  • modified proteins e.g. , derivitized L-arginine, lysine, serine, tyrosine (with the latter two examples having undergone hydroxy modification) or the like, provide targeted nitric oxide species for an extended period of time, thereby achieving longer duration target site-specific agonist effects upon the nitric oxide pathway.
  • the present invention also provides methods of modulating a nitric oxide pathway to achieve a therapeutic effect in a target cell population.
  • Such methods include administration of a therapeutically effective amount of an agent including a targeting portion capable of delivering the agent to a target site and an effector portion capable of modulating the nitric oxide pathway at the target site.
  • Another embodiment of the present invention involves methods of modulating nitric oxide synthase.
  • Methods of the present invention additionally or alternatively include administration of a free molecule to achieve systemic nitric oxide pathway or nitric oxide synthase modulation.
  • the present invention contemplates a methodology involving administration of a targeting portion-ligand (e.g.. avidin or streptavidin) molecule followed by administration of a anti-ligand (e.g.. biotin) -effector portion (e.g..).
  • nitric oxide donor moiety nitric oxide donor moiety
  • the target cell population is "pre-targeted" by the targeting portion-ligand moiety to achieve enhanced localization of the anti-ligand- effector portion to those sites.
  • Other methods of the present invention involve administration of nitric oxide synthase directly or indirectly bound to a targeting portion and subsequent administration of a nitric oxide synthase modulator.
  • Therapeutic effects which can be achieved-by methods of the present invention are those that involve a constitutive or an inducible target cell nitric oxide pathway.
  • Exemplary therapeutic effects include treatment of restenosis, treatment of homeostasis of endothelial cells, cancer treatment and treatment of septic shock.
  • Fig. 1 is a schematic representation of the nitric oxide pathway exhibited by vascular smooth muscle cells.
  • Fig. 2 is a schematic representation of a two- step therapeutic method embodiment of the present invention for restenosis treatment.
  • Fig. 3A graphically depicts experimental data regarding protein synthesis inhibition capability of Calphostin C with respect to vascular smooth muscle cells.
  • Fig. 3B graphically depicts experimental data regarding protein synthesis inhibition capability of methyl arginine with respect to vascular smooth muscle cells.
  • Fig. 3C graphically depicts experimental data regarding protein synthesis inhibition capability of nitro arginine with respect to vascular smooth muscle cells.
  • Fig. 3D graphically depicts experimental data regarding protein synthesis inhibition capability of nitroglycerin with respect to vascular smooth muscle cells.
  • Fig. 4A graphically depicts experimental data regarding DNA synthesis inhibition capability of sodium nitroprusside with respect to vascular smooth muscle cells.
  • Fig. 4B graphically depicts experimental data regarding DNA synthesis inhibition capability of nitroglycerin with respect to vascular smooth muscle cells.
  • Targeting Portion A molecule that binds to a defined population of cells.
  • the targeting portion may bind a receptor, an enzymatic substrate, an antigenic determinant or other binding site present on the target cell population.
  • Proteins, such as antibodies and other polypeptides, are used throughout the specification as prototypical examples of targeting portions of the present invention.
  • Effector Portion A molecule or combination of molecules capable of modulating a constitutive or an inducible nitric oxide pathway.
  • Analog A molecule that is structurally similar to and exhibits the opposite modulating activity from the molecule of which it is an "analog.”
  • Ljgand/Anti-Ligand Pair A member of a complementary/anti-complementary set of molecules that demonstrate specific binding, generally of relatively high affinity.
  • Exemplary ligand/anti-ligand pairs include hapten/antibody, ligand/receptor and biotin/avidin or streptavidin. Biotin/avidin is used throughout the specification as a prototypical example of a ligand/anti- ligand pair.
  • Ligand A relatively small, soluble molecule that exhibits rapid serum, blood and/or whole body clearance when administered intravenously in an animal or human.
  • Anti-Ligand A moiety that demonstrates high affinity, and, preferably, multivalent binding of the complementary ligand. More preferably, the anti-ligand is large enough to avoid rapid renal clearance, and contains sufficient multivalency to accomplish crosslinking and aggregation of targeting portion-ligand conjugates.
  • Avidin A compound capable of high affinity, multivalent or univalent binding of biotin, including avidin, streptavidin and derivatives and analogs thereof.
  • the present invention is directed to targeted agents capable of modulating a nitric oxide pathway to achieve a therapeutic effect.
  • An illustrative nitric oxide pathway is shown in Fig. 1, with the reaction being susceptible to cytokine induction and L-arginine analog inhibition.
  • Nitric oxide synthesis from L-arginine is modulated by nitric oxide synthase, an enzyme for which a plurality of isozymes have been identified.
  • the presence of cytokines at nitric oxide synthesis sites (intercellular or intracellular sites where the reactants and the catalyst are found) therefore results in nitric oxide formation, while the presence of N-alkyl (as opposed to N-hydroxy) L-arginine analogs prevents such formation.
  • a preferred embodiment of a targeted agent of the present invention includes a targeting portion bound to an effector portion by covalent or non-covalent binding, with covalent binding preferred.
  • Equivalent targeted agents of this embodiment of the present invention are fusion proteins including targeting and effector moieties.
  • the targeting portion delivers the targeted agent to target cell sites, while the effector portion modulates a nitric oxide pathway at such sites.
  • the targeting portion and the effector portion may be formed as separate molecules or be combined in a single molecule.
  • Targeting portions useful in the present invention include molecules capable of binding a defined population of cells, which cells exhibit a constitutive or an inducible nitric oxide pathway. Targeting portions which facilitate agent internalization into the target cells are preferred for effector portions requiring metabolic processing to impact the level of nitric oxide in the cell. For effector portions that spontaneously decompose to nitric oxide (i.e.. do not require metabolic processing to achieve an increased nitric oxide level) , internalization capability is of less significance, because the highly lipophilic and diffusible nitric oxide molecule can reach intracellular effector sites.
  • Preferred targeting portions for use in the practice of the present invention include moieties capable of localizing to smooth muscle cells such as vascular smooth muscle cells, corpus cavernosum smooth muscle cells or the like, brain cells such as cerebellar cells, certain neurons or the like, liver cells such as hepatocytes and Kupffer cells or the like, cancer cells including melanoma, lymphoma, adenocarcinoma, hepatoma or the like, endothelial cells, macrophages, platelets, fibroblasts, esangial cells, neutrophils, renal collecting duct cells, intestinal cells such as intestinal muscle cells, intestinal neural cells or the like, chondrocytes or the like, for example.
  • smooth muscle cells such as vascular smooth muscle cells, corpus cavernosum smooth muscle cells or the like
  • brain cells such as cerebellar cells, certain neurons or the like
  • liver cells such as hepatocytes and Kupffer cells or the like
  • cancer cells including melanoma, lymphoma,
  • targeting portions are antibodies or fragments thereof that localize to such target cells, avidin or streptavidin conjugates of such antibodies or fragments, receptor specific proteins or peptides, wherein such targeting portions can optionally be used in liposome, microparticle, nanoparticle or other appropriate dosage forms.
  • Effector portions of the present invention include molecules or combinations of molecules that are capable of modulating a nitric oxide pathway of cells to which the targeting portion of the targeted agent localizes.
  • modulation encompasses both enhancement and inhibition of nitric oxide production or of reactions modulated by nitric oxide.
  • Preferred effector moieties useful in the practice of the present invention include nitrate compounds, L-arginine, poly-L-arginine, arginine analogs (e.g..
  • glutamic acid glutamic acid, poly- glutamic acid, N-methyl-D-aspartate, poly-N-methyl-D- aspartate) , glucocorticosteroids, corticosteroids, transforming growth factors, macrophage deactivating factor, cytokines such as interferon, tumor necrosis factor, interleukin-1, interleukin-2 and lipopolysaccharide, aminoguanidine, heme, non-heme iron or compounds which facilitate site-specific delivery of molecular oxygen or superoxide anion, superoxide ion scavengers, GMP phosphodiesterase inhibitors, histamine, vasopressin, epinephrine, serotonin, acetylcholine (ACh) , ADP, alpha- adrenergic agonists, thrombin and bradykinin, for example.
  • cytokines such as interferon, tumor necrosis factor, interleukin-1, inter
  • Fig. 1 is illustrative of a typical nitric oxide synthase-mediated reaction.
  • An inducer/potentiator of nitric oxide synthase production or activity causes an increase in production of nitric oxide (Fig. 1) or products of other reactions mediated by nitric oxide synthase.
  • an inhibitor of nitric oxide synthase production or activity causes a decrease in the production of nitric oxide (Fig. 1) or other nitric oxide synthase-mediated reaction product.
  • Embodiments of this aspect of the present invention involve a targeting portion bound to an effector portion by covalent or non-covalent binding, with covalent binding preferred.
  • Equivalent targeted agents of this embodiment of the present invention are fusion proteins including targeting portions and effector portions.
  • the targeting portion of such agents of the present invention delivers the agent to target cell sites, while the effector portion modulates a nitric oxide synthase catalyzed reaction at such sites.
  • Targeting portions useful in this embodiment of the present invention include molecules capable of binding a defined population of cells, which cells exhibit receptor- mediated reactions in which nitric oxide synthase plays a role. Targeting portions which facilitate agent internalization into the target cells are amenable to the practice of the present invention. Also, targeting portions specific for extracellular epitopes are useful in the practice of the present invention, as a result of the highly diffusible nature of nitric oxide.
  • Preferred targeting portions for use in the practice of this embodiment of the present invention include moieties capable of localizing to smooth muscle cells such as vascular smooth muscle cells, corpus cavernosum smooth muscle cells or the like, brain cells such as cerebellar cells, certain neurons or the like, liver cells such as hepatocytes, Kupffer cells or the like, certain cancer cells including melanoma, lymphoma, adenocarcinoma, hepatoma or the like, endothelial cells, macrophages, platelets, fibroblasts, mesangial cells neutrophils, polymorphonucleargranulocytes, renal collecting duct cells, intestinal cells such as intestinal muscle cells and intestinal neural cells, chondrocytes, or the like.
  • smooth muscle cells such as vascular smooth muscle cells, corpus cavernosum smooth muscle cells or the like
  • brain cells such as cerebellar cells, certain neurons or the like
  • liver cells such as hepatocytes, Kupffer cells or the like
  • cancer cells including melanom
  • targeting moieties are antibodies or fragments thereof that localize to such target cells, avidin or streptavidin conjugates of such antibodies or fragments, receptor specific proteins or peptides, optionally delivered in liposome, microparticle nanoparticle or other appropriate dosage forms.
  • Effector portions of this embodiment of the present invention include molecules or combinations of molecules that are capable of modulating nitric oxide synthase in cells to which the targeting portion of the targeted agent localizes.
  • modulation encompasses both enhancement and inhibition of nitric oxide synthase production or activity.
  • Preferred effector moieties useful in the practice of this embodiment of the present invention include glucocorticosteroids, L-arginine modified with imidazole, acetylenic or allylic functionalities, L-arginine, poly-L- arginine, L-arginine analogs (e.g.
  • excitatory amino acids and polymers thereof e.g. , glutamic acid, poly-glutamic acid, N-methyl- D-aspartate, poly-N-methyl-D-aspartate
  • aminoguanidine for example.
  • L-arginine and poly-L-arginine exhibit potentiating effects
  • modified L-arginine, L-arginine analogs, aminoguanidine and glucocorticosteroids are exemplary of moieties that exhibit inhibitory effects.
  • effector molecules useful in targeted agents of the present invention exhibit reversible effector properties, while others exhibit irreversible effector properties. More specifically, "reversible” and “irreversible” effector moieties may exhibit either a potentiating or an inhibitory effect on the target cell. Administration of an oppositely acting effector molecule results in reversal of the effect exerted by a reversible effector. In contrast, the metabolic modulation of an irreversible effector is not reversed by subsequent administration of an oppositely acting effector moiety.
  • exemplary reversible effector molecules are modified arginine, such as arginine substituted with functionalities including imidazole, acetylene, cyclopropyl as well as nitro.
  • Exemplary irreversible effector molecules are N-methyl arginine, N-allyl arginine and the like.
  • arginine modified with an imidazole functionality may be targeted by an appropriate antibody and internalized within a target cell.
  • the modified arginine exhibits an inhibitory effect on nitric oxide synthase.
  • L-arginine for example, which has a potentiating effect on nitric oxide synthase
  • the inhibitory effect of the imidazole-modified arginine is reversed. If the first administered targeted agent inhibitory effector moiety is allyl arginine or methyl arginine subsequent administration of L-arginine will not appreciably reverse the inhibitory effect, however.
  • effector portion useful in targeted agents of the present invention includes a molecule which reacts with or otherwise deactivates nitric oxide. Such effector portions have an antagonistic effect on nitric oxide pathways.
  • exemplary effector portions of this embodiment of the present invention are heme, non-heme iron, compounds that facilitate the site-specific delivery of molecular oxygen, superoxide ion, or reducing equivalents such as thiols, ascorbate and the like.
  • Targeting portions and effector portions of targeted agents of the present invention may be bound by any convenient mechanism therefor.
  • the link between the targeting portion and the effector portion is cleavable through the action of intracellular processes or in response to the intracellular environment for embodiments of the present invention involving effector portions that require metabolic processing to impact the target cellular nitric oxide level.
  • effector portions that spontaneously decompose to nitric oxide i.e. , do not require metabolic processing to achieve an increased nitric oxide level
  • internalization capability is of less significance, because the highly lipophilic and diffusible nitric oxide molecule can reach intracellular effector sites.
  • proteinaceous targeting portions may be directly linked to amino acid-based effector portions through a peptide bond by procedures that are known in the art.
  • Peptide bonds are cleavable by endogenous peptidases of target cells, thereby releasing the effector molecule from the agent in those cells.
  • such moieties may be directly linked by other covalent or non-covalent bonds or indirectly linked through cross-linkers such as heterobifunctional cross- linking compounds, ligand-anti-ligand pairs or the like.
  • cross-linkers such as heterobifunctional cross- linking compounds, ligand-anti-ligand pairs or the like.
  • Both the indirect and direct coupling methods employ reactive groups, such as hydroxyl, amino, a ido, or sulf ydryl groups, in an effector molecule and in a corresponding targeting portion as well as in the optional cross-linking agent. Bonds, such as a peptide bond, disulfide bond, thioester bond, amide bond, thioether bond, or the like, can be employed for this purpose.
  • Targeting portions and effector portions of targeted agents of the present invention may be formed using fusion protein genetic engineering technology. Fusion proteins are hybrid proteins generated using recombinant DNA technology. A fusion protein is translated from messenger RNA as one continuous polypeptide chain, with the protein or peptide components joined together by peptide bonds. A recombinant DNA fusion sequence corresponding to a conjugate of the claimed invention may be cloned and expressed according to standard procedures.
  • the recombinant DNA fusion sequence is inserted . in vitro into an expression vector capable of replication in a particular host microorganism.
  • the expression vector is derived from a plasmid or virus. See Old and Primrose, Principals of Gene Manipulation. 2d ed. , University of California Press, pp. 104-17, 1981; PCT Patent Application Publication No. WO 86/00528; United States Patent Nos. 4,599,311 and 4,704,362; and British Patent No. GB 2,119,804.
  • Another aspect of the present invention involves administration of a free (i.e.. untargeted or unbound to a targeting portion) effector molecule to achieve systemic modulation of a nitric oxide pathway or nitric oxide synthase.
  • exemplary effector molecules useful in the practice of this aspect of the present invention include arginine, arginine analogs such as N G -monomethyl-L-arginine, N G -nitro-L-arginine, N G -amino-L-arginine, N G -cyclopropyl-L- arginine, N G -amino-L-homoarginine, N G -nitro-L-arginine methyl ester or the like, nitrate compounds such as sodium nitroprusside, nitrosothiols, nonoates and nitroglycerin and amyl nitrate, isosorbide diuate, hydralazine and penta- erythritol
  • Such free effector molecule administration may be conducted alone or, as preferred, in combination with a targeting portion-effector portion targeted agent of the present invention.
  • Free effector/targeted agent administration can be serial or simultaneous. In this manner, both targeted and systemic modulation is achieved.
  • nitric oxide donor moieties spontaneously degrade under physiological conditions to form nitric oxide.
  • Such moieties include molecules that have undergone sulfhydryl modification to form S-nitroso substituted molecules, L-arginine modification to form nonoate equivalents or the like.
  • Sulfhydryl modification may be accomplished, for example, by disulfide bond reduction to form thiol functionalities or by utilization of endogenous thiol groups on the agent.
  • one or more targeting portion thiol groups are derivitized with nitric oxide gas or NaN0 2 to yield a moiety of the following structure: protein-S-NO.
  • cysteine forms such a moiety.
  • protein-O-nitroso e.g.. tyrosine or serine derivitization
  • protein-N-nitroso e.g. , lysine derivitization
  • Such modified moieties may act as both targeting portions and effector portions and, therefore, as targeted agents in accordance with the present invention.
  • S- nitroso substituted antibodies for example, such modification may be conducted upon endogenous sulfhydryls or sulfhydryls introduced into the antibody structure by the exposure of the antibody to a reducing agent. Provided the modification does not interfere with the target cell binding, S-nitroso substituted antibodies constitute targeted agents of the present invention.
  • S-nitroso moieties may be introduced into a physiologically acceptable sulfhydryl containing molecule to generate an effector portion capable of delivery by a targeting portion.
  • Nonoate groups can be introduced into L-arginine, L-lysine, serine, tyrosine or like molecules to generate an effector portion capable of delivery by a targeting portion.
  • the modified molecules or nitric oxide donor containing effector portion targeted agents deliver a nitric oxide producing species (through degradation of the donor moiety) at the target site for an extended time period (e.g. , from about 1 hour to about 24 hours) . In this manner, sustained agonist effects upon the target nitric oxide pathway are achieved.
  • An example of this inventive concept involves the targeting of endothelial cells which exhibit a constitutive nitric oxide pathway. Elevated levels of nitric oxide cause vasodilation. At lower levels, nitric oxide serves to inhibit platelet activation and neutrophil adherence and to combat the cytotoxic effects of superoxide radicals. Ischemia and reperfusion interfere with proper endothelial cell function with respect to nitric oxide production. Addition of S-nitroso compounds or targeted S-nitroso effector molecules, for example, provides a source of nitric oxide to the target cell, thereby protecting ischemic- reperfused endothelial tissues from the action of superoxide radicals.
  • Exemplary targeting portions useful in the practice of this embodiment of the present invention are S- nitroso or nonoate modified endothelial cell targeting antibodies or proteins or endothelial targeting antibodies or proteins bound to S-nitroso or nonoate containing effector molecules.
  • Additional embodiments of the present invention provide targeting portion-ligand and effector portion-anti- ligand conjugates or fusion proteins for serial administration to a mammalian recipient.
  • Targeting portion- ligand is administered first to permit localization to the target site or removal from the recipient by the relevant metabolic pathway therefor.
  • Effector portion-anti-ligand is subsequently administered (with a time lag between administrations ranging from about 4 hours to about 72 hours) .
  • the effector portion-anti-ligand is either bound at the target site by ligand-anti-ligand interaction with the targeting portion-ligand or is rapidly cleared from the recipient (with a clearance time ranging from about 5 minutes to about 2 hours) .
  • Preferred targeting moiety structures include antibodies, proteins or peptides, or dosage forms such as liposomes or solid supports (e.g. , polymers) capable of specifically localizing to a target cell population.
  • Preferred effector portions useful in these embodiments of the present invention are nitric oxide donor molecules or an isozyme of the nitric oxide synthase enzyme.
  • an avidinylated monoclonal antibody for example, an avidinylated monoclonal antibody
  • biotinylated nitric oxide donor molecule is administered, with an exemplary molecule being of the following structure:
  • X is a spacer group selected to impart certain desired physical or biological properties to the biotinylated effector molecule.
  • exemplary X groups are -HN-(CH 2 ) n ⁇ NH-, -RN-(CH 2 ) n -NH- (where R is an alkyl group), spermine, multicarboxylate compounds (e.g.. diethylene triamine penta- acetic acid, often abbreviated as DTPA) , dextran or the like, which impart sustained release of nitric oxide when targeted or rapid clearance properties when not localized to target cells.
  • n ranges from 1 to about 5.
  • X is NH, O or S.
  • X is NH, O or S.
  • Another example of this inventive concept involves avidin-targeting portion administration followed by administration of biotinylated nitric oxide synthase.
  • This embodiment of the present invention is also practicable by a nitric oxide synthase effector portion/targeting portion conjugate, fusion protein or other dosage form.
  • These targeted agents of the present invention localize nitric oxide synthase enzymatic activity at a target site following administration, for example, by the preferred injection or implantation routes.
  • nitric oxide release by this procedure is not characterized by the adverse systemic consequences observed for moieties such as drugs or toxins, because of the lipophilicity and reactivity of nitric oxide. More specifically, nitric oxide is a highly lipophilic molecule that easily diffuses into cells located adjacent to or in the vicinity of the targeting moiety. In addition, nitric oxide is highly reactive and, therefore, does not appreciably diffuse into the systemic circulation prior to being inactivated.
  • a preferred ligand-anti-ligand pair useful in the practice of the present invention is avidin-biotin. Either avidin or biotin can constitute the ligand or anti-ligand of the agents of the present invention, with biotin being preferred as the anti-ligand.
  • Ligand-targeting protein conjugates, fusion proteins and other dosage forms may be prepared in accordance with known coupling, genetic engineering and related techniques. Anti-ligand-effector portion conjugates, fusion proteins and other dosage forms may also be prepared using known techniques.
  • exemplary target cell types are smooth muscle cells such as vascular smooth muscle cells, corpus cavernosum smooth muscle cells or the like, brain cells such as cerebellar cells, certain neurons or the like, liver cells such as hepatocytes, Kupffer cells or the like, certain cancer cells including melanoma, lymphoma, adenocarcinoma, hepatoma or the like, endothelial cells, macrophages, platelets, fibroblasts, mesangial cells neutrophils, polymorphonucleargranulocytes, renal collecting duct cells, intestinal cells such as intestinal muscle cells and intestinal neural cells, chondrocytes or the like.
  • smooth muscle cells such as vascular smooth muscle cells, corpus cavernosum smooth muscle cells or the like
  • brain cells such as cerebellar cells, certain neurons or the like
  • liver cells such as hepatocytes, Kupffer cells or the like
  • cancer cells including melanoma, lymphoma, adenocarcinoma, hepatom
  • cell types exhibiting receptor-mediated reactions in which nitric oxide synthase plays a role are susceptible to the practice of the present invention.
  • Exemplary cell types are vascular smooth muscle cells, brain cells, liver cells, certain cancer cells, endothelial cells and the like.
  • Cells exhibiting nitric oxide synthase controlled, NMDA receptor mediated events, for example, are amenable to the practice of the present invention.
  • vascular smooth muscle cells employ a metabolic pathway to produce nitric oxide, a molecule which is capable of activating soluble guanylate cyclase and the cGMP cascade.
  • This nitric oxide pathway is schematically shown in Fig. 1.
  • Other cell types are impacted by cGMP modulation through manipulation of a nitric oxide pathway.
  • platelet aggregation is inhibited by elevated cGMP levels (corresponding to elevated nitric oxide levels) .
  • Na absorption of renal duct collecting cells is inhibited by elevated cGMP (nitric oxide) levels.
  • nitric oxide synthesis is induced by cytokines, such as tumor necrosis factor and interleukin-1. Consequently, such cytokines are effector portions useful in the practice of the present invention to modulate the nitric oxide pathway of vascular smooth muscle cells to produce nitric oxide.
  • cytokines such as tumor necrosis factor and interleukin-1. Consequently, such cytokines are effector portions useful in the practice of the present invention to modulate the nitric oxide pathway of vascular smooth muscle cells to produce nitric oxide.
  • the lack of nitric oxide has been implicated as a contributor to the proliferation of arterial smooth muscle cells that is characteristic of restenosis. Stimulation of nitric oxide synthesis diminishes or obviates the problem of nitric oxide deficiency, thereby promoting homeostasis of smooth muscle cells.
  • nitric oxide synthase is competitively inhibited by L-arginine analogs, such as N G -monomethyl-L- arginine, N G -nitro-L-arginine, N G -amino-L-arginine, N G - cyclopropyl-L-arginine, N G -amino-L-homoarginine, N G -nitro- L-arginine methyl ester or the like. Consequently, cytokine- induced effects can be inhibited by L-arginine analogs as well. This fact forms one of the bases for the two-step vascular smooth muscle treatment protocol shown in Fig. 2.
  • nitric oxide synthase activation and/or action of nitric oxide synthase is inhibited by an administered antibody-nitric oxide synthase inhibitor conjugate (e.g. , an L-arginine analog-antibody conjugate) .
  • an administered antibody-nitric oxide synthase inhibitor conjugate e.g. , an L-arginine analog-antibody conjugate
  • production of nitric oxide is decreased, thereby inhibiting nitric oxide- potentiated events such as cGMP cascade activation, platelet disaggregation, smooth muscle relaxation, and cytotoxicity.
  • a monoclonal antibody-nitric oxide synthase inducer conjugate e.g..
  • step 2 is conducted with a time lag ranging from about 5 minutes to about 72 hours following step 1.
  • Monoclonal antibody targeting portions useful in the practice of this embodiment of the present invention localize to chondroitin sulfate proteoglycans (CSPGs) synthesized by vascular smooth muscle cells and pericytes.
  • CSPGs chondroitin sulfate proteoglycans
  • An exemplary targeting portion useful in this aspect of the present invention involves a NR-AN-01 monoclonal antibody (a subculture of NR-ML-05) .
  • the monoclonal antibody designated NR-ML-05 binds a 250kD CSPG synthesized by melanoma cells (Morgan et al., U.S. Patent No. 4,897,255) . Smooth muscle cells and pericytes also synthesize a 250kD CSPG as well as other CSPGs.
  • NR-ML-05 binding to smooth muscle cells has been disclosed (Fritzberg et al., U.S. Patent No. 4,879,225).
  • Monoclonal antibody NR-ML-05 and subculture NR-ML-05 No. 85- 41-4I-A2, freeze # A2106, have both been deposited with the American Type Culture Collection, Rockville, MD and granted Accession Nos. HB-5350 and HB-9350, respectively.
  • NR-ML- 05 is the parent of, and structurally and functionally equivalent to, subclone NR-AN-01, disclosed herein.
  • the amino acid residues involved in the multi-point kinetic association of the NR-AN-01 monoclonal antibody with a CSPG marker antigenic epitope are deter inable by computer-assisted molecular modeling and by the use of mutants having altered antibody binding affinity.
  • the binding-site amino acids and three dimensional model of the NR-AN-01 antigen binding site serve as a molecular model for constructing functional equivalents, e.g.
  • minimal polypeptides short polypeptides that have binding affinity for a CSPG epitope synthesized by vascular smooth muscle cells and pericytes.
  • minimal polypeptide refers to a sequence of at least six amino acids in length.
  • Three-dimensional modeling is also useful to construct other functional equivalents that mimic the binding of NR-AN-01 to its antigenic epitope, e.g. , "mimetic" chemical compounds that mimic the three-dimensional aspects of NR-AN-01 binding to its epitope in a CSPG target antigen.
  • mietic refers to an organic chemical polymer constructed to achieve the proper spacing for binding to the amino acids of an NR-AN-01 CSPG target synthesized by vascular smooth muscle cells or pericytes.
  • Murine monoclonal antibodies or "humanized” murine antibody are also useful as targeting portions in accordance with the present invention.
  • murine monoclonal antibody may be "humanized” by genetically recombining nucleotide sequence encoding the murine Fv region (i.e. , containing the antigen binding sites) or the complementarity determining regions thereof with the nucleotide sequence encoding a human constant domain region and an Fc region, e.g. , in a manner similar to that disclosed in European Patent Application No. 0,411,893 A2.
  • Humanized targeting portions are recognized to decrease the immunoreactivity of the antibody or polypeptide in the host recipient, permitting an increase in the half-life and a reduction in the possibility of adverse immune reactions.
  • Targeted agents of the present invention may be administered in any convenient manner therefor.
  • an infusion catheter may be employed to deliver agents to certain target sites, such as vascular smooth muscle cells.
  • Other routes of administration also find utility in the practice of the present invention. Exemplary administration routes are injection by the intravenous, intralymphatic, intrathecal, or other intracavity routes, oral administration, implantation and the like, depending primarily upon the target site and the structure of the targeted agent.
  • Targeted agents of the present invention are administered in such amounts as to deliver a therapeutically effective amount of effector molecule to target sites.
  • Appropriate administered doses depend on a variety of factors that are largely patient specific.
  • the components of the targeted agent used also impact dose amounts in ways that are known to or routinely ascertainable by practitioners in the art.
  • targeted agent is administered to mammals at a dose ranging between 1.0 mg and 1.0 g, depending upon the physiological characteristics of the patient and the ailment involved.
  • a practitioner in the art is capable of identifying an appropriate dose and administration route for a given recipient with a given ailment.
  • Certain cancer cell types are characterized by or cause an accumulation of effector cells, such as macrophages, in the vicinity thereof.
  • effector cells employ a nitric oxide pathway.
  • Nitric oxide has been observed to have tumor suppressive properties. Consequently, effector cell nitric oxide production is manipulable by administration of targeted agents of the present invention directed to macrophage target cells. Increased target cell nitric oxide production caused by the targeted delivery of a nitric oxide synthesis potentiating effector portion results in a tumor cell suppression therapeutic benefit.
  • nitric oxide donor containing targeted agents or modified molecules provide a sustained release source of nitric oxide or a nitric oxide synthesis potentiator (e.g.. a cytokine) to tumor cell sites.
  • exemplary targeting portions useful in this aspect of the present invention include antibodies, peptides, proteins and the like directed to tumor cell antigens.
  • Minimal peptides, mimetic organic chemical compounds, human or humanized monoclonal antibodies and the like that localize to cancer cells are also useful in accordance with the present invention. Such moieties may be identified and constructed or isolated in accordance with known techniques.
  • An exemplary targeting portion useful in the practice of the present invention is NR-LU-10, a monoclonal antibody reactive with a pancarcinoma glycoprotein of approximately 40,000 dalton molecular weight.
  • Nitric oxide is present in elevated amounts in septic patients exhibiting hemodynamic compromise. Production of nitric oxide results in reduced vascular contractility. Inhibition of nitric oxide production reverses the hypotension.
  • useful effector portions for this embodiment of the present invention modulate the nitric oxide pathway in a manner opposite to that indicated above for cancer treatment. More specifically, antagonist rather than agonist effector molecule function is utilized. Consequently, L-arginine analogs are exemplary effector molecules useful in the practice of this aspect of the present invention.
  • Exemplary targeting portions useful in this embodiment of the present invention include NR-AN-01 or an endothelial cell targeting portion.
  • Targeting portions useful in this aspect of the present invention include antibodies, peptides, proteins and the like.
  • Minimal peptides, mimetic organic chemical compounds, human or humanized monoclonal antibodies and the like that localize to endothelial cells are also useful in accordance with the present invention.
  • Nitric oxide is a mediator of inflammation. Chronic, non-infectious inflammation results in the stimulation of nitric oxide production. Consequently, modulation of nitric oxide at sites of inflammation impacts the operation of the inflammation cascade. Effector moieties capable of either stimulation or inhibition of nitric oxide production find utility in the practice of the present invention to, respectively, augment or diminish the activity of the inflammation cascade. Exemplary effector moieties are
  • Targeting portions useful in the practice of this embodiment of the present invention include those directed to antigens that are up regulated during the inflammation process such as adhesion antigens.
  • hepatic cells can be protected from inflammation related injury by enhanced nitric oxide synthesis, thereby rendering targeting portions that localize to such cells useful in the practice of the present invention.
  • Exemplary targeting portions are galactosyl proteins (which target hepatocytes) and mannosyl proteins, for example.
  • Targeting portions useful in this aspect of the present invention include antibodies, peptides, proteins and the like.
  • Minimal peptides, mimetic organic chemical compounds, human or humanized monoclonal antibodies and the like that localize to relevant target cell populations are also useful in accordance with the present invention.
  • nitric oxide exerts protective effects on organs such as the liver. Some of these effects may be the result of nitric oxide interaction with superoxide ion or prevention of microvascular perfusion deficits.
  • the liver exhibits an inducible nitric oxide pathway where maximum induction of nitric oxide synthesis is accomplished by multiple inducing moieties (e.g.. interferon, interleukin- 1, tumor necrosis factor and lipopolysaccharide) .
  • inducing moieties e.g. interferon, interleukin- 1, tumor necrosis factor and lipopolysaccharide
  • cytokines or other effector moieties capable of inducing nitric oxide production in hepatic cells are preferred for use in liver therapy embodiments of the present invention.
  • Targeting portions of agents used in this aspect of the present invention localize to hepatocytes. Exemplary of such targeting portions are galactosyl proteins, for example.
  • Targeting portions useful in this aspect of the present invention include antibodies, peptides, proteins and the like.
  • Minimal peptides, mimetic organic chemical compounds, human or humanized monoclonal antibodies and the like that localize to relevant target cell population are also useful in accordance with the present invention.
  • acrophages Like hepatocytes, acrophages exhibit a cytokine- inducible nitric oxide synthase. Macrophages process the antigens of intercellular pathogens and present such antigens to T-cells for recognition and cell-mediated destruction. Nitric oxide produced by macrophages modulates proliferative and cytolytic responses of T-cells. The presence of nitric oxide suppresses T-cell proliferation and cytolytic processes mediated by T-cells. Consequently, L-arginine analogs such as N G -monoomethyl-L-arginine can be targeted to macrophages to down modulate nitric oxide synthesis if T-cell proliferative or cytolytic activity is desired.
  • L-arginine analogs such as N G -monoomethyl-L-arginine can be targeted to macrophages to down modulate nitric oxide synthesis if T-cell proliferative or cytolytic activity is desired.
  • T-cell targeting agents useful in the practice of the present invention are monoclonal antibodies, fragments thereof and other specific receptor binding moieties.
  • Minimal peptides, mimetic organic chemical compounds, human or humanized monoclonal antibodies and the like that localize to relevant target cell population are also useful in accordance with the present invention.
  • Glutamate has been implicated in neurodegeneration disorders such as ischemic hypoxic insults, Alzheimer's disease and Huntington's disease. Glutamate neurotoxicity has been found to be mediated by nitric oxide. Inhibition of nitric oxide production by, for example, L-arginine analogs such as N G -nitro-L-arginine and N G -monomethyl-L- arginine results in a decrease in glutamate receptor mediated cell death.
  • a major source of the nitric oxide that mediates neurotoxicity is nitric oxide synthase containing neurons. Such neurons are therefore appropriate targets for localization by the targeting portions of this aspect of the present invention. Exemplary targeting portions are monoclonal antibodies, fragments thereof and other specific receptor binding moieties. Minimal peptides, mimetic organic chemical compounds, human or humanized monoclonal antibodies and the like that localize to relevant target-cell population are also useful in accordance with the present invention.
  • nitric oxide production is inhibited by agents that generate the superoxide ion.
  • Superoxide ion scavengers such as superoxide dismutase, ascorbate, glutathione or the like, may be employed as effector molecules in targeted agents of the present invention designed to prevent or diminish superoxide inhibition of the nitric oxide pathway.
  • Targeting portions useful in the practice of this embodiment of the present invention localize to cells which exhibit a constitutive or inducible nitric oxide pathway and are exposed to agents generating oxide radicals such as the superoxide radical.
  • Endothelial cells, smooth muscle cells, macrophages, brain cells and the like are exemplary sets of target cells.
  • Exemplary targeting agents therefore include monoclonal antibodies, fragments thereof and other specific receptor binding moieties.
  • Minimal peptides, mimetic organic chemical compounds, human or humanized monoclonal antibodies and the like that localize to relevant target cell population are also useful in accordance with the present invention.
  • Hyperaemia thought to limit acid injury to the mucosa caused by acid back diffusion through a disrupted gastric ucosal barrier, is an increase in gastric mucosal blood flow.
  • Nitric oxide mediates gastric mucosal vasodilation.
  • An increase in nitric oxide production results in an increase in mucosal blood flow. This phenomena is believed to be the result of the operation of the nitric oxide pathway of neurotransmitters responsible for submucosal and mucosal vasodilation. Consequently, L-arginine effector molecules or other nitric oxide synthesis potentiators may be targeted to such neurotransmitters by an appropriate targeting portion.
  • Exemplary targeting portions are monoclonal antibodies, fragments thereof and other specific receptor binding moieties.
  • Minimal peptides, mimetic organic chemical compounds, human or humanized monoclonal antibodies and the like that localize to relevant target cell population are also useful in accordance with the present invention.
  • Nitric oxide is a mediator of relaxation of corpus cavernosu resulting from nonadrenergic noncholinergic neurotransmission. The presence of nitric oxide therefore promotes penile erection.
  • Nitric oxide synthesis potentiators, such as L-arginine can be employed as effector portions of agents of the present invention designed to achieve penile erection.
  • Corpus cavernosum smooth muscle cells are the appropriate target for the targeting portion used in this embodiment of the present invention.
  • Exemplary targeting portions are monoclonal antibodies, fragments thereof and other specific receptor binding moieties.
  • Minimal peptides, mimetic organic chemical compounds, human or humanized monoclonal antibodies and the like that localize to relevant target cell population are also useful in accordance with the present invention.
  • Vascular smooth muscle cells (B054 baboon smooth muscle cells) at 40,000 cells/ml were seeded in sterile 24 well plates at 1 ml/well. The plates were incubated overnight at 37 ° C, C0 2 , 95% air in a humidified atmosphere (saturation) . Log dilutions of the therapeutic agent being tested were incubated with the vascular smooth muscle cells for 5 minutes or 24 hours. Samples of the therapeutic agent were diluted in DMEM: F- 12 media ( hittaker Bioproducts, alkersville, Maryland) with 5% fetal bovine serum (FBS, Gibco) and 5% Serum Plush ® (JRH Biological) .
  • FBS hittaker Bioproducts, alkersville, Maryland
  • Serum Plush ® JRH Biological
  • Vascular smooth muscle cells were incubated in complete media with 5% FBS (Gibco) overnight at 37 ° C in a humidified, 5% C0 2 environment in sterile 24 well plates.
  • the media was aspirated from the wells and serum free media supplemented with growth factors (DMEM: F-12 basal media supplemented with growth factor cocktail, catalog number 11884, which contains insulin (5 micrograms/ml) , transferrin (5 micrograms/ml) and sodium selenite (5 nanograms/ml) , available from Sigma Chemical, St. Louis, Missouri) was added. Cells were incubated in this media for 24 hours.
  • DMEM F-12 basal media supplemented with growth factor cocktail, catalog number 11884, which contains insulin (5 micrograms/ml) , transferrin (5 micrograms/ml) and sodium selenite (5 nanograms/ml) , available from Sigma Chemical, St. Louis, Missouri
  • Vascular smooth muscle cells were seeded at 4.0 x 10* cells/ml media/well on a commercially prepared four well slide (Nunc, Inc. , Naperville, Illinois) . Enough slides were seeded to accommodate two pulsed exposure lengths (5 minutes and 24 hours) and prescribed increment evaluation points (24 hours to 128 hours) . All slides were run in duplicate to reveal any assay anomalies. Nitroglycerin was diluted in the same media used in the 3 H-leucine and 3 H- thymidine assays.
  • the media was aspirated and subsequently replaced with 1 ml of 10% neutral buffered formal in for one hour to allow for proper fixation.
  • These fixed slides were stained by hematoxylin (nuclear) and eosin (cytoplasmic) for morphologic evaluation and grading.
  • Figs. 3A-3D and 4A-4B show exemplary results of 3 H-leucine and 3 H-thymidine studies for vascular smooth muscle target cells (B054 cells) exposed to various effector moieties.
  • Fig. 3A indicates that Calphostin C inhibits protein synthesis of the target cell population
  • Figs. 3B-3D indicate that methyl arginine, nitro arginine and nitroglycerin do not inhibit protein synthesis. Consequently, administration of Calphostin C will kill the target cells, while administration of the other effector portions will not.
  • Figs. 4A and 4B indicate that sodium nitroprusside and nitroglycerin inhibit DNA synthesis. Consequently, administration of such moieties will result in target cell metabolic modulation (diminished proliferation) .
  • a comparison of Figs. 3D and 4B indicates that nitroglycerin administration results in metabolic modulation of the target cells without killing the target cells.

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Abstract

La présente invention se rapporte à des agents ciblés pouvant moduler une voie d'oxyde nitrique ou la synthase d'oxyde nitrique afin de produire un effet thérapeutique. Certains agents ciblés comprennent une partie de ciblage pouvant apporter un agent vers un site cible et une partie effecteur pouvant moduler une voie d'oxyde nitrique ou une synthase d'oxyde nitrique au niveau du site cible. La présente invention se rapporte également à des procédés de modulation d'une voie d'oxyde nitrique ou d'une synthase d'oxyde nitrique afin de produire un effet thérapeutique dans une population de cellules cibles.
PCT/US1994/000894 1993-01-28 1994-01-26 Modulation ciblee de synthase d'oxyde nitrique ou de voie d'oxyde nitrique WO1994016729A1 (fr)

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Cited By (24)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2726186A1 (fr) * 1994-10-28 1996-05-03 Centre Nat Rech Scient Medicaments contenant des inhibiteurs de la nitrique-oxyde synthase
WO1996016645A1 (fr) * 1994-11-25 1996-06-06 The Wellcome Foundation Limited Utilisation de donneurs d'oxyde nitrique dans le domaine medical
US5594032A (en) * 1994-11-10 1997-01-14 Gonzalez-Cadavid; Nestor F. Amelioration of human erectile dysfunction by treatment with iNOS, inducers of iNOS or iNOS cDNA
EP0871376A1 (fr) * 1995-11-09 1998-10-21 The Board Of Trustees Of The Leland Stanford Junior University Amelioration de la fonction vasculaire par modulation de la production ou de l'activite de l'oxyde nitrique endogene
US5854234A (en) * 1993-10-21 1998-12-29 G. D. Searle & Co. Amidino dervatives useful as nitric oxide synthase inhibitors
US5883251A (en) * 1995-04-20 1999-03-16 G. D. Searle & Co. Azepine derivatives useful as nitric oxide synthase inhibitors
US5904938A (en) * 1995-02-16 1999-05-18 The General Hospital Corporation Treatment of vascular thrombosis and restenosis with inhaled nitric oxide
US5908842A (en) * 1995-12-08 1999-06-01 Merck & Co., Inc. Substituted 2-acylamino-pyridines as inhibitors of nitric oxide synthase
US5945408A (en) * 1996-03-06 1999-08-31 G.D. Searle & Co. Hydroxyanidino derivatives useful as nitric oxide synthase inhibitors
US5958958A (en) * 1997-07-22 1999-09-28 G.D. Searle & Co. 1,2,4-oxa diazolino and 1,24-oxa diazolidion heterocycles as useful nitric oxide synthase inhibitors
EP1015033A2 (fr) * 1997-01-10 2000-07-05 AltaRex, Corp. Perylenequinones substituees destinees a la therapie photodynamique
US6090839A (en) * 1996-12-23 2000-07-18 Merck & Co., Inc. Antidiabetic agents
US6160000A (en) * 1996-12-23 2000-12-12 Merck & Co., Inc. Antidiabetic agents based on aryl and heteroarylacetic acids
US6344473B1 (en) 2000-08-07 2002-02-05 G.D. Searle & Co. Imidazoles useful as nitric oxide synthase inhibitors
US6489323B1 (en) 1998-06-10 2002-12-03 G.D. Searle & Co. Heterobicyclic and tricyclic nitric oxide synthase inhibitors
US6747063B2 (en) 1996-04-23 2004-06-08 Cellegy Pharmaceuticals, Inc. Combination therapy for treatment of erectile dysfunction
US7452916B2 (en) 1993-06-11 2008-11-18 The Board Of Trustees Of The Leland Stanford Junior University Enhancement of vascular function by modulation of endogenous nitric oxide production or activity
WO2017214339A1 (fr) 2016-06-08 2017-12-14 Abbvie Inc. Anticorps anti-b7-h3 et conjugués anticorps-médicaments
WO2017214456A1 (fr) 2016-06-08 2017-12-14 Abbvie Inc. Anticorps anti-cd98 et conjugués anticorps-médicament
WO2017214322A1 (fr) 2016-06-08 2017-12-14 Abbvie Inc. Anticorps anti-b7-h3 et conjugués anticorps-médicaments
WO2018195302A1 (fr) 2017-04-19 2018-10-25 Bluefin Biomedicine, Inc. Anticorps anti-vtcn1 et conjugués anticorps-médicament
US10640563B2 (en) 2016-06-08 2020-05-05 Abbvie Inc. Anti-B7-H3 antibodies and antibody drug conjugates
EP4218929A1 (fr) 2014-03-21 2023-08-02 AbbVie Inc. Anticorps anti-egfr et conjugués anticorps-médicament
US11759527B2 (en) 2021-01-20 2023-09-19 Abbvie Inc. Anti-EGFR antibody-drug conjugates

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4863713A (en) * 1986-06-23 1989-09-05 The Board Of Trustees Of Leland Stanford Jr. Univ. Method and system for administering therapeutic and diagnostic agents
US4867973A (en) * 1984-08-31 1989-09-19 Cytogen Corporation Antibody-therapeutic agent conjugates
US4981979A (en) * 1987-09-10 1991-01-01 Neorx Corporation Immunoconjugates joined by thioether bonds having reduced toxicity and improved selectivity

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4867973A (en) * 1984-08-31 1989-09-19 Cytogen Corporation Antibody-therapeutic agent conjugates
US4863713A (en) * 1986-06-23 1989-09-05 The Board Of Trustees Of Leland Stanford Jr. Univ. Method and system for administering therapeutic and diagnostic agents
US4981979A (en) * 1987-09-10 1991-01-01 Neorx Corporation Immunoconjugates joined by thioether bonds having reduced toxicity and improved selectivity

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
FASEB JOURNAL, Volume 3, issued January 1989, L.J. IGNARRO, "Endothelium-Derived Nitric Oxide: Actions and Properties", pages 31-36. *
TIBS, Volume 14, issued December 1989, M.A. MARLETTA, "Nitric Oxide: Biosynthesis and Biological Significance", pages 488-492. *

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* Cited by examiner, † Cited by third party
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US7452916B2 (en) 1993-06-11 2008-11-18 The Board Of Trustees Of The Leland Stanford Junior University Enhancement of vascular function by modulation of endogenous nitric oxide production or activity
US6046211A (en) * 1993-10-21 2000-04-04 G.D. Searle & Co. Amidino derivatives useful as nitric oxide synthase inhibitors
US6071906A (en) * 1993-10-21 2000-06-06 G. D. Searle & Co. Imidino piperidine derivatives useful as nitric oxide synthase inhibitors
US6448286B1 (en) 1993-10-21 2002-09-10 G.D. Searle & Co. Imino pyrrolidine derivatives useful as nitric oxide synthase inhibitors
US5854234A (en) * 1993-10-21 1998-12-29 G. D. Searle & Co. Amidino dervatives useful as nitric oxide synthase inhibitors
FR2726186A1 (fr) * 1994-10-28 1996-05-03 Centre Nat Rech Scient Medicaments contenant des inhibiteurs de la nitrique-oxyde synthase
WO1996013256A1 (fr) * 1994-10-28 1996-05-09 Centre National De La Recherche Scientifique (Cnrs) Medicaments contenant des inhibiteurs de la nitrique-oxyde synthase pour le traitement des pathologies d'immunodeficience
US5594032A (en) * 1994-11-10 1997-01-14 Gonzalez-Cadavid; Nestor F. Amelioration of human erectile dysfunction by treatment with iNOS, inducers of iNOS or iNOS cDNA
WO1996016645A1 (fr) * 1994-11-25 1996-06-06 The Wellcome Foundation Limited Utilisation de donneurs d'oxyde nitrique dans le domaine medical
US5904938A (en) * 1995-02-16 1999-05-18 The General Hospital Corporation Treatment of vascular thrombosis and restenosis with inhaled nitric oxide
US6063407A (en) * 1995-02-16 2000-05-16 The General Hospital Corporation Treatment of vascular thrombosis and restenosis with inhaled nitric oxide
US5883251A (en) * 1995-04-20 1999-03-16 G. D. Searle & Co. Azepine derivatives useful as nitric oxide synthase inhibitors
US6043261A (en) * 1995-04-20 2000-03-28 G. D. Searle & Co. Pyrrolodino imidines useful as nitric oxide synthase inhibitors
EP0871376A4 (fr) * 1995-11-09 2004-03-24 Univ Leland Stanford Junior Amelioration de la fonction vasculaire par modulation de la production ou de l'activite de l'oxyde nitrique endogene
EP0871376A1 (fr) * 1995-11-09 1998-10-21 The Board Of Trustees Of The Leland Stanford Junior University Amelioration de la fonction vasculaire par modulation de la production ou de l'activite de l'oxyde nitrique endogene
US5908842A (en) * 1995-12-08 1999-06-01 Merck & Co., Inc. Substituted 2-acylamino-pyridines as inhibitors of nitric oxide synthase
US5945408A (en) * 1996-03-06 1999-08-31 G.D. Searle & Co. Hydroxyanidino derivatives useful as nitric oxide synthase inhibitors
US6747063B2 (en) 1996-04-23 2004-06-08 Cellegy Pharmaceuticals, Inc. Combination therapy for treatment of erectile dysfunction
US6090839A (en) * 1996-12-23 2000-07-18 Merck & Co., Inc. Antidiabetic agents
US6160000A (en) * 1996-12-23 2000-12-12 Merck & Co., Inc. Antidiabetic agents based on aryl and heteroarylacetic acids
EP1015033A2 (fr) * 1997-01-10 2000-07-05 AltaRex, Corp. Perylenequinones substituees destinees a la therapie photodynamique
EP1015033A4 (fr) * 1997-01-10 2000-07-05 Altarex Inc Perylenequinones substituees destinees a la therapie photodynamique
US5958958A (en) * 1997-07-22 1999-09-28 G.D. Searle & Co. 1,2,4-oxa diazolino and 1,24-oxa diazolidion heterocycles as useful nitric oxide synthase inhibitors
US5981556A (en) * 1997-07-22 1999-11-09 G.D. Searle & Co. 1,3-diazolino and 1,3-diazolidino heterocycles as useful nitric oxide synthase inhibitors
US6136829A (en) * 1997-07-22 2000-10-24 G.D. Searle & Co. Oxathiadiazole derivatives usful as iNOS inhibitors
US6489323B1 (en) 1998-06-10 2002-12-03 G.D. Searle & Co. Heterobicyclic and tricyclic nitric oxide synthase inhibitors
US6344473B1 (en) 2000-08-07 2002-02-05 G.D. Searle & Co. Imidazoles useful as nitric oxide synthase inhibitors
EP4218929A1 (fr) 2014-03-21 2023-08-02 AbbVie Inc. Anticorps anti-egfr et conjugués anticorps-médicament
WO2017214339A1 (fr) 2016-06-08 2017-12-14 Abbvie Inc. Anticorps anti-b7-h3 et conjugués anticorps-médicaments
WO2017214456A1 (fr) 2016-06-08 2017-12-14 Abbvie Inc. Anticorps anti-cd98 et conjugués anticorps-médicament
WO2017214322A1 (fr) 2016-06-08 2017-12-14 Abbvie Inc. Anticorps anti-b7-h3 et conjugués anticorps-médicaments
US10640563B2 (en) 2016-06-08 2020-05-05 Abbvie Inc. Anti-B7-H3 antibodies and antibody drug conjugates
WO2018195302A1 (fr) 2017-04-19 2018-10-25 Bluefin Biomedicine, Inc. Anticorps anti-vtcn1 et conjugués anticorps-médicament
US11759527B2 (en) 2021-01-20 2023-09-19 Abbvie Inc. Anti-EGFR antibody-drug conjugates

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