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WO1993025239A1 - Derives d'arabinogalactane et leurs utilisations - Google Patents

Derives d'arabinogalactane et leurs utilisations Download PDF

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Publication number
WO1993025239A1
WO1993025239A1 PCT/US1992/005091 US9205091W WO9325239A1 WO 1993025239 A1 WO1993025239 A1 WO 1993025239A1 US 9205091 W US9205091 W US 9205091W WO 9325239 A1 WO9325239 A1 WO 9325239A1
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Prior art keywords
arabinogalactan
complex
agent
cell receptor
therapeutic agent
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PCT/US1992/005091
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English (en)
Inventor
Chu Jung
Philip Enriquez
Stephen Palmacci
Lee Josephson
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Advanced Magnetics Inc.
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Application filed by Advanced Magnetics Inc. filed Critical Advanced Magnetics Inc.
Priority to CA002135295A priority Critical patent/CA2135295A1/fr
Priority to PCT/US1992/005091 priority patent/WO1993025239A1/fr
Priority to EP92914217A priority patent/EP0646018A1/fr
Priority to JP6501422A priority patent/JPH07507794A/ja
Publication of WO1993025239A1 publication Critical patent/WO1993025239A1/fr
Priority to NO944838A priority patent/NO944838L/no

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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
    • 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
    • A61K47/59—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 obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/605—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 obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the macromolecule containing phosphorus in the main chain, e.g. poly-phosphazene
    • 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
    • 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
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/645—Polycationic or polyanionic oligopeptides, polypeptides or polyamino acids, e.g. polylysine, polyarginine, polyglutamic acid or peptide TAT

Definitions

  • This invention relates to the synthesis and methods of use of therapeutic agents targeted to cells, especially hepatocytes.
  • the safety and efficacy of a therapeutic agent is a function of (i) its intrinsic biological activity and (ii) the biodistribution achieved after its administration.
  • Many potentially useful therapeutic agents possess a biochemical activity ameliorating a particular pathological condition, but the presence of the agent in normal, nonpathological tissue results in deleterious effects that prevent the use of the agent. Damage to a normally functioning kidney, bone marrow, liver tissue or other organ may limit the use of therapeutic agents with established antiviral activity, or agents with established anti-cancer activity.
  • Targeting is the modification of a therapeutic agent so that after injection or oral administration the uptake by a specific population of cells is increased relative to uptake of the unmodified agent.
  • a therapeutic agent is a compound administered with the intent of changing in a beneficial manner some physiological function.
  • Therapeutic agents include radioprotective agents, chemoprotective agents, antiviral agents, antibodies, enzymes, and peptides.
  • One method of targeting therapeutic agents to specific cells involves attaching them to carrier molecules recognized by receptors performing receptor mediated endocytosis.
  • Of particular interest is targeting via the asialoglycoprotein receptor of hepatocytes. This receptor is present in high levels on normal hepatocytes but in lower levels or not at all on transformed hepatocytes (hepatoma cells) .
  • Diagnostic and therapeutic agents have been attached to asialoglycoprotein carriers and neoglycoprotein carriers recognized by the asialoglycoprotein receptor and targeted to the cells, see Table II of Meijer and van der Sullies, Pharm. Res. (1989) 6:105-118 and Ranade, J. Clin. Pharmacol. (1989) 29:685-694. Molecules recognizing the asialoglycoprotein receptor are most often either asialoglycoproteins or neoglycoproteins.
  • Asialoglycoproteins are formed by removing the sialic acid of glycoproteins and exposing galactose residues.
  • Neoglycoproteins are formed by attaching multiple galactose residues to non-glycoproteins such as human albumin.
  • targeting can be achieved only if the affinity of the carrier for the receptor is maintained.
  • the differential reactivity of the protein amine and carbohydrate hydroxyl groups of glycoprotein carriers, e.g. asialofetuin, is commonly used to achieve this goal.
  • the highly reactive amine groups of protein lysine residues are selectively modified, while the hydroxyl groups of carbohydrate are left intact and continue to recognize the receptor. Examples of this strategy are given in Van der Sluijs et al. (above) and in "Liver Diseases, Targeted Diagnosis and Therapy Using Specific Receptors and Ligands" (1991) Ed. G.Y. Wu and C.H.
  • Glycoproteins are prepared from animal cells and insuring nonconta ination with human infective viral pathogens is a major issue.
  • Glycoproteins will not generally tolerate organic solvents during conjugate synthesis, because such solvents frequently lead to a loss of biological activity and denaturation.
  • Glycoproteins can be toxic and/or antigenic.
  • Arabinogalactans are a class of polysaccharides obtained from the cell walls of many species of trees and other plants. A common, commercially available source of arabinogalactan is the American Western larch (Larix occidentalis) . Arabinogalactan from this source is used as a binder, emulsifier or stabilizer in foods.
  • arabinogalactans consists of a largely 1-3 linked D-galactose backbone with 1-6 linked branch chains of L-arabinoses and D-galactoses at practically every residue on the backbone.
  • the ratio of galactose to arabinose is between 5 to 1 and 10 to 1
  • arabinogalactans from plant sources in general range from about 1 to 4 to about 10 to 1
  • arabinogalactans from plant sources in general range from about 1 to 4 to about 10 to 1
  • arabinogalactans have different molecular weights with values of about 1-2 million to about 10,000 dalton ⁇ [Blake, J.D., Clarke, M.L.
  • Arabinogalactan sulfate has been used to form salts with drugs to influence drug absorption and prolong drug action [US4609640]. Acidic forms of arabinogalactan occur naturally having a composition which includes uranic acid [Clarke, A.E., Anderson, R.L., Stone, B.A. ,
  • the present invention provides for derivatives of arabinogalactan which can be used to target therapeutic agents to the cells possessing the asialoglycoprotein receptor.
  • polysaccharide arabinogalactan to target therapeutic agents to cells via the asialoglycoprotein receptor, a feature of the current invention, overcomes problems encountered when glycoproteins are used for this purpose.
  • arabinogalactan is a polysaccharide, it will tolerate exposure to organic solvents, which normally denature proteins during conjugate synthesis. Composed exclusively of sugars, the polysaccharide presents a narrow spectrum of reactive sites, an advantage compared to proteins where the variety of reactive sites can lead to unwanted synthetic byproducts. This advantage is evident in the examples below.
  • Arabinogalactan has low toxicity and antigenicity.
  • Arabinogalactan in its natural form reacts with the asialoglycoprotein receptor. This helps reduce manufacturing cost because the deasialylation reaction normally used to expose the penultimate galactose of glycoproteins is avoided.
  • arabinogalactan can be modified in a number of ways to produce molecules which preserve the useful affinity for the asialoglycoprotein receptor. This is surprising since arabinogalactan does not afford protein or amino groups for selective modifications distal from the receptor binding site. The ability to modify arabinogalactan while retaining its biological activity permits its use as a carrier for a wide variety of therapeutic agents with various targeting strategies.
  • targeting may be employed to deliver a therapeutic agent to normal rather than pathological tissue. This strategy is employed when it is desirable to protect normal tissues from other generally toxic agents; in some cases agents of known but controlled toxicity are employed in therapy.
  • the targeting of protective agents used in conjunction with normally toxic radiation, as in radiation therapy is an embodiment of the current invention and example of this type of targeting.
  • the targeting of protective agents used with chemotherapeutic agents used in cancer treatment is another embodiment of the current invention.
  • arabinogalactan derivatives of the invention must interact strongly with the asialoglycoprotein receptor, so they can be used to target therapeutic agents to cells via that receptor.
  • An assay to determine the strength of the interaction of arabinogalactan derivatives of the invention with the receptor is presented.
  • the antiviral therapeutic agent adenosine arabinoside mono-5'-phosphate is coupled to arabinogalactan.
  • ARA-AMP adenosine arabinoside mono-5'-phosphate
  • both antiviral therapeutic agents may also separately be coupled to arabino-galactans.
  • the radioprotective agent S-2-(3 aminopropylamino) ethyl- thiophosphoric acid (known as WR2721) is attached to arabinogalactan.
  • the invention provides methods and compositions which enable the attachment of a variety of therapeutic agents to arabinogalactan and the delivery of those agents into the cytoplasm of cells via endocytotic activity of the asialoglycoprotein receptor.
  • arabinogalactan used here in a preferred embodiment is highly purified and substantially free of endotoxins, and is derived from the Western Larch and has a single peak by size exclusion chromatography of about 20,000 daltons.
  • Arabinogalactan can be used in its native, 20,000 dalton form; alternatively polymers of arabinogalactan (molecular weight greater than the 20,000 dalton form), or degradative products (molecular weight below the 20,000 dalton form) can be used.
  • Purified arabinogalactan has a single peak of 20,000 daltons by gel filtration, and a ratio of galactose to arabinose of 5 to 1 as determined by the alditol acetate method.
  • arabinogalactan is distinguishable from other polysaccharides including dextrans, starches, celluloses, inulins, 1-4 linked galactan and gum arabic. Though chemically distinguishable from arabinogalactan, gum arabic is another polysaccharide which like arabinogalactan interacts with the asialoglycoprotein receptor.
  • the present invention provides for conjugates of arabinogalactan with the therapeutic agents such as ARA-AMP or WR2721.
  • the present invention also provides derivatives of arabinogalactan which interact with the asialoglycoprotein receptor.
  • a therapeutic agent can be targeted into the cells possessing that receptor, chiefly the hepatocytes.
  • Asialoglycoprotein receptors are dramatically reduced in primary hepatocellular cancers, and totally absent in secondary cancers to the liver, but are found in high concentration on normal hepatocytes [Josephson, Gro an et al., Mag. Res. Imag. (1990) 8:637-646].
  • Hepatocytes are the predominant cell possessing this receptor, and endocytose a large proportion of injected radiolabelled asialoglycoproteins [Hubbard, Wilson et. al., J. Cell Biol. (1979) 83:47-64].
  • asialoglycoprotein receptors have been detected on Kupffer cells [Lee, Haekyung, et al. Biol. Chem. Hoppe-Seyler (1988) 369: 705-714], bone marrow cells [Samoloski and Daynes, Proc. Nat. Acad. Sci. (1985) 82:2508-2512] and rat testis [Abullah and Kierszenbaum, J. Cell Biol. (1989) 108: 367-375].
  • a therapeutic agent may be targeted to any asialoglycoprotein receptor positive cell.
  • any receptor positive cell including stem cells, may be protected with a receptor targeted radioprotective agent based on arabinogalactan.
  • ARA-AMP is an antiviral therapeutic agent that has been evaluated in the treatment of hepatitis B, though its use is associated with serious neurological side effects [Lok, A.S., Wilson, L.A. et al., J. Antimicrob. Chemother. (1984) 11: 93-9; Hoffnagel, J.H. et al. , J. Hepatol. (1986) 3: S73-80] .
  • ARA-AMP conjugated to arabinogalactan, and targeted to asialoglycoprotein receptor possessing cells where viral replication is ongoing is expected to reduce unwanted side effects by reducing the concentration of the drug in the central nervous system and increasing the concentration of the drug in the organ of viral replication.
  • ARA-AMP has been coupled to a glycoprotein recognized by the asialoglycoprotein receptor [US 4794170].
  • Other antiviral therapeutic agents which may be used for this purpose include acyclovir and Ara-A.
  • antibodies may include polyclonal antibodies, monoclonal antibodies or antibody fragments.
  • the natural occurrence of antibodies in serum reflect past exposure to a virus but may have little or no protective, activity because viral replication occurs within the cytoplasm of cells [I.M. Roitt, "Essential Immunology,” Blackwell Scientific, London (1991), p. 28].
  • hepatitis B virus replication occurs within the hepatocytes of the liver and antibodies to viral antigens cannot directly bind the virus during this replication.
  • an antibody to a hepatitis B viral protein is conjugated to arabinogalactan, it will be targeted via the asialoglycoprotein receptor to the cytoplasm of hepatocytes. Within the cytoplasm the antiviral antibody will bind replicating hepatitis B virus and become an effective therapeutic agent.
  • arabinogalactan derivatives described have no known pharmacological activity, other than their ability to bind the receptor, but provide a substrate for attaching therapeutic agents, e.g., attachment to the amino, carboxyl, sulfhydryl, phosphoryl or other functional groups of the derivative.
  • the resulting conjugate will target the therapeutic agent to cells possessing the asialoglycoprotein receptor, principally the hepatocytes of the liver.
  • the carboxyl groups afforded by the succinyl-arabinogalactan, glutaryl-arabinogalactan and DTPA-arabinogalactan conjugates can be used to attach molecules through the use of carbonii ides or other agents.
  • the amino groups afforded by the arabinogalactan hydrazide (Example 3) or poly-L-lysine arabinogalactan (Examples 6, 8) can also be used to attach therapeutic agents by a variety of reactions.
  • the strong positive charge of poly-L-lysine can cause some agents such as negatively charged nucleic acids to adhere by ionic exchange forces [Wu, G.Y. and Wu, C.H., J. Biol Chem. (1987) 262: 4429-2232].
  • a preferred embodiment of this invention is a composition comprising arabinogalactan and poly-L-lysine, wherein the intended use is as a carrier for genes or antisense oligonucleotides used in parenteral administration [Degols, G.
  • galactose oxidase treatment of arabinogalactan can be used to create aldehyde groups.
  • the aldehyde groups can be reacted with diamino compounds (e.g. ethylenediamine) , to form a Schiff base, followed by reduction with sodium borohydride.
  • the resulting amino derivative of arabinogalactan can then be used for the attachment of therapeutic agents.
  • WR2721 has been the subject of recent clinical studies to ascertain whether it can be used to protect the normal cells of cancer patients during radiotherapy [Kligerman, M.M. , Liu, T. , Liu, Y.
  • WR2721 as a chemoprotectant has been objected to based on the lack of evidence that it selectively protects normal cells; i.e. it may protect normal and cancer cells from radiation [The Pink Sheet, Feb 3, 1992, 54, #5].
  • the attachment of WR2721 to arabinogalactan will overcome this shortcoming, directing the agent to cells possessing the asialoglycoprotein receptors.
  • the radioprotective activity of WR2721 will be targeted to normal cells since the asialoglycoprotein receptor is found chiefly on non-cancerous hepatocytes, see above.
  • Free radical scavengers other than WR2721 can be attached to arabinogalactan, and targeted to receptor bearing cells.
  • These scavengers include melanins [Hill, H.Z., Huselton, C. , Pilas, B. , Hill, G.J. ; Pigment Cell Res (1987) 1: 81-6], Trolox [Wu, T.W. , Hashimoto, N. , Au, J.X. , Wu, J., Mickle, D.A. , Carey, D. , Hepatology (1991) 13.: 575- 80], cysteamine derivatives [Schor, N.F., Siuda, J.F., Lomis, T.J. , Cheng, B. , Biochem J (1990) 267: 291-6], cationic aminothiols generally, glutathiols, and vitamin E derivatives.
  • the interaction of the arabinogalactan derivative with the asialoglycoprotein receptor can be determined in vivo.
  • the ability of a derivative to interact with the asialoglycoprotein receptor is assessed by its ability to block the clearance of a substance recognized to interact with the asialoglycoprotein receptor based on earlier work.
  • An arabinogalactan coated superparamagnetic iron oxide colloid interacts with this receptor and a quantitative assay for its clearance has been described below.
  • the arabinogalactan coated superparamagnetic iron oxide is rapidly cleared via the asialoglycoprotein receptor with a blood half-life of 2.8 minutes.
  • the interaction of free arabinogalactan with the asialoglycoprotein receptor effects an increase in blood half-life of this substance, providing a basis for evaluating the blocking ability of arabinogalactan derivatives.
  • a Sprague-Dawley rat (200-300 grams) is anesthetized (100 mg/kg of Inactin) and injected with a defined dose of a blocking agent, followed by an arabinogalactan coated superparamagnetic iron oxide at 40 umoles Fe/kg.
  • Blood is withdrawn and 1/T1, the spin-spin relaxation rate, determined.
  • the enhancement in 1/T1 is directly proportional to the concentration of superparamagnetic iron oxide, and from changes in 1/T1 the blood half-life is determined as described [Josephson et al. Mag Res. Imag. (1990) 8: 637-646].
  • Table 1 indicates that arabinogalactan can tolerate a substantial degree of modification produced by many different types of reactions, without losing its activity as a blocking agent (receptor binding activity) .
  • covalent modification especially high levels of covalent modification, generally decreases or destroys biological function.
  • arabinogalactan tolerates random modification with excellent retention of its receptor-recognizing biological activity.
  • two derivatives tested, the phosphoryl arabinogalactan and succinyl-arabinogalactan were more potent as blocking agents than the parent arabinogalactan. The basis for this highly surprising improved reactivity is unknown.
  • lactose a disaccharide-containing galactose, is substantially less active a blocker than arabinogalactan.
  • the ability of a derivatization procedure to damage the binding affinity of arabinogalactan for the asialoglycoprotein receptor is shown by example 18.
  • the acetate derivative has greater than 5 milli-equivalents of acetate per gram of arabinogalactan acetate and exhibited substantially reduced blocking activity.
  • arabinogalactan can be modified by the addition of phosphoryl, sulfhydryl, amino, carboxyl, halo, or acylimidazol groups, with receptor binding activity being unaffected.
  • the initial modification is performed on the hydroxyl groups on the arabinogalactan.
  • the derivatives can be used to prepare conjugates with therapeutic agents, as for example arabinogalactan-WR2721 or arabinogalactan-AMP (Table 1) . In some cases we describe the preparation of amino or carboxy arabinogalactan derivatives with no known therapeutic activity.
  • These derivatives can be used to attach a wide range of drugs or ligands to the amino or carboxy groups of derivatized arabinogalactan, with generally known crosslinking and conjugation chemistries. These derivatives can also be used to attach macromolecules like genes, proteins, antibodies and enzymes to arabinogalactan. A recent compendium of applicable reactions is S.W. Wong, "Chemistry of Protein Conjugation and Cross-linking," CRC Press Boca Raton, 1991). Reagents used to couple proteins to solid phase amino or carboxyl groups can also be used after minor modifications (see I. Chibata, "Immobilized Enzymes," Halstead Press, New York 1978) . Some examples of therapeutic agents that can be conjugated to arabinogalactan to provide useful pharmaceutical agents are listed in Table 2.
  • Example 1 Bro ination of arabinogalactan.
  • arabinogalactan (AG) used is from the Western Larch and chromatographs produce a single peak of about 20,000 daltons by size exclusion chromatography.
  • Example 2 Treatment of arabinogalactan with sodium borohydride. Ten grams sodium borohydride is added to 3,500 grams of a 28.6% (w/w) solution of arabinogalactan. The mixture is stirred overnight, and then dialyzed for six days against 35 liters of water (changing the water daily) using 3,500 dalton cut-off dialysis tubing to remove unreacted NaBH 4 . The 3-methyl-2-benzothiazolone hydrazone test for aldehyde is used to compare the aldehyde content of the arabinogalactan starting material to sodium borohydride reduced arabinogalactan. Arabinogalactan showed the blue dye formation characteristic of aldehyde, reduced arabinogalactan produced no dye, indicating essentially complete reduction.
  • Example 3 Hydrazino-arabinogalactan.
  • Example 2 Ten grams of reduced arabinogalactan (Example 2) is dissolved in 35 ml of a 7.1% (w/v) aqueous solution of Zn(BF ) 2 . Fifty ml of epibromohydrin is added and the solution stirred for 90 minutes at 100°C. The brominated arabinogalactan is precipitated in 150 ml cold (4°C) acetone, redissolved in water, and precipitated in 150 ml cold ethanol. Five grams of this brominated arabinogalactan product is dissolved in 15 ml of 0.3 M aqueous borate, pH 8. Ten grams hydrazine is added and the mixture is stirred for 24 hours at room temperature.
  • hydrazido-arabinogalactan is precipitated in 150 ml cold (4 ⁇ C) acetone, redissolved in water and precipitated in 150 ml cold ethanol.
  • the product hydrazide content is analyzed by acid-base titration and showed 0.25 milliequivalents hydrazide per gram of product.
  • Example 4 Arabinogalactan conjugated to adenosine 5' monophosphate (AMP)
  • adenosine 5'-monophosphate AMP
  • Arabinogalactan-hydrazide 0.6 g, example 2
  • One gram (5.2 mmoles) of l-ethyl-3,4- dimethylaminopropyl) carbodiimide is added and the reaction maintained at room temperature for 64 hours.
  • the product is purified by ultrafiltration using an Amicon YM3 ultrafilter, further purified by precipitation from ethanol. A yield of 323 mg of product was obtained.
  • the product was analyzed by cation exchange chromatography (Rainin Synchropak, strong cation exchange So 300 A, 25 x 0.5 cm column) using a buffer of 0.1 mM, pH 7.0 phosphate buffer at flow rate 0.5 ml/min) .
  • a single broad peak at 5.7 minutes with no evidence for underivatized AMP (retention time 6.3 minutes) was observed.
  • the number of AMP molecules per gram of AG- AMP product based on the comparison of HPLC area under the curve monitoring at 260 nm is 0.24, indicating approximately a 95% conversion of available hydrazide groups.
  • the UV/VIS spectrum of the AG-AMP product is virtually identical to underivatized AMP.
  • the analysis of product by size exclusion (Amicon Cellufine GC200M) chromatography shows a molecular weight approximately equivalent to underivatized arabinogalactan, about 20,000 daltons.
  • AG-AMP The activity of AG-AMP was evaluated in the animal model as described above. 150 mg/kg of this substance was an effective blocker of the superparamagnetic iron-oxide colloid, extending the half-life of the colloid to greater than 100 minutes (Table 1) .
  • Example 5 Arabinogalactan conjugated to adenine arabinoside 5' monophosphate (ARA-AMP) .
  • ARA-AMP adenine arabinoside 5' monophosphate
  • adenine arabinoside 5'- monophosphate ARA-AMP
  • adenine arabinoside 5'- monophosphate ARA-AMP
  • adenine arabinoside 5'- monophosphate ARA-AMP
  • 0.6 grams of arabinogalactan-hydrazide is then added and the pH adjusted to 7.5 with the addition of sodium hydroxide.
  • One gram (5.2 mg) of l-ethyl-3,4- dimethylaminopropyl) carbodiimide is added and the reaction maintained at room temperature for 64 hours.
  • the product is purified by ultrafiltration using an Amicon YM3 ultrafilter, and then further purified by precipitation from ethanol and dried, yielding 280 mg of product.
  • Poly(L) lysine hydrochloride (1,000-4,000 daltons, 0.5 grams) is dissolved in 2 ml borate buffer (0.2M) and the pH adjusted to 9.0 with sodium hydroxide. 100 mg arabinogalactan and 50 mg sodium cyanoborohydride is added, and the reaction heated for 24 hours at 50°C. The product mixture is purified using an Amicon YM3 ultrafilter. The retentate containing the polylysine-arabinogalactan conjugate showed a positive ninhydrin test for amine and positive anthrone test for polysaccharide. The yield was 30 mg.
  • Example 8 Poly(L)lysine-arabinogalactan (prepared from acylimidazole-arabinogalactan) .
  • acylimidazole-arabinogalactan (Example 7) and 0.2 grams poly(L)lysine (1,000-4,000 daltons) is dissolved in 5 ml of 0.2M borate buffer and the pH adjusted to 8.6 with sodium hydroxide. The reaction is allowed to proceed for 24 hours at 5°C. The product is isolated first by precipitation in ethanol, and then purified using an Amicon YM10 ultrafilter. The retentate shows a positive test for amine and carbohydrate using the ninhydrin and anthrone tests, respectively, while the final filtrate is negative for amine. The yield is 310 mg.
  • poly(L)lysyl-arabinogalactan is analyzed by cation exchange chromatography HPLC (Rainin Synchropak strong cation exchange resin, So 300A, 25 X 0.5 cm), using pH 5.5, 25 mM phosphate buffer at a 1 ml/min flow rate.
  • Poly(L)lysine bound to arabinogalactan is verified by its UV spectrum.
  • Example 9 Phosphoryl-arabinogalactan.
  • the product showed 0.21 milli-equivalents of phosphate per gram of product both by acid base titration and by colorimetric quantitation of inorganic phosphate (inorganic phosphorus kit, Sigma Chemical, St.Louis, MO) following trifluoroacetic acid hydrolysis (2M acid for 1 hour at 120 ⁇ C) .
  • inorganic phosphate inorganic phosphorus kit, Sigma Chemical, St.Louis, MO
  • the activity of phosphorylated arabinogalactan was evaluated in the animal model as described above. 150 mg/kg of this substance was an effective blocker of the superparamagnetic iron-oxide colloid, extending the half- life of the colloid to greater than 51 minutes (Table 1) .
  • Example 10 Treatment of arabinogalactan with galactose oxidase (GO) .
  • Example 14 S-2-(3 aminopropylamino) ethyl-thiophosphate- dextran-arabinogalactan from thiophosphorylated dextran. Polythiophosphorylation of dextran. Ten grams of dextran is suspended in 60 ml of anhydrous pyridine. The suspension is cooled in an ice water bath. To the cooled suspension is added dropwise with stirring 10 ml (98.4 mmoles) of thiophosphoryl chloride. Once the addition is complete the reaction mixture is allowed to warm to room temperature with constant stirring. The reaction flask is then immersed in an oil bath and heated for 16 hours at 40°C.
  • the slightly yellow colored reaction mixture is cooled in an ice bath. Once cooled, water is added slowly dropwise while the reaction suspension is vigorously stirred. After about 10 ml of water has been added to the reaction mixture a solution of 1 N NaOH is added until a pH of 9.5 is reached. The solution is then evaporated at room temperature to an oil. The residue is mixed with 20 ml of water, which results in a clear homogeneous solution. This solution is added dropwise to 200 ml of 0°C ethanol which is vigorously stirred. The resulting white precipitate is collected on a coarse fritted funnel and dried under vacuum. Titration with 0.5 M hydrochloric acid indicates that 1 mmole of thiophosphate is incorporated per gram of polysaccharide.
  • Reduced arabinogalactan is brominated as described in Example 3. 2 grams of this brominated arabinogalactan is added to 1 gram of WR2721 in 10 ml of 0.2M borate and the pH adjusted to 8.0. The mixture is stirred for 16 hours at room temperature. Arabinogalactan-WR2721 is purified by Amicon YM3 ultrafiltration, then precipitated in acetone and redissolved in water. Finally it is precipitated in ethanol and dried. The final product is dissolved in 0.1 N HC1 and titrated with 0.1 N NaOH.
  • the arabinogalactan-WR2721 final product was shown to have 0.66 milli-equivalents of WR2721 per gram of product.
  • the product analyzed by size exclusion chromatography shows the major component has a molecular weight of about 25,000 daltons.
  • arabinogalactan-WR2721 The activity of arabinogalactan-WR2721 was evaluated in the animal model as described above. Injection of 150 mg/kg of this substance was an effective blocker of the superparamagnetic iron-oxide colloid clearance, extending the half-life of the colloid to 86 minutes (Table 1) .
  • Example 16 Arabinogalactan-WR272l from phosphorylated arabinogalactan.
  • Arabinogalactan-phosphate (8 grams, example 9), 1.2 grams 1-ethyl -(3,4-dimethylaminopropyl)carbodiimide, and 1 gram of WR2721 are mixed together in 20 ml of water. The pH is adjusted to 7.5 with the addition of sodium hydroxide, and the mixture allowed to stand in the dark at room temperature for approximately 64 hours.
  • the product, WR2721 linked to arabinogalactan through its primary amine esterified to the phosphate on arabinogalactan-phosphate is purified by ultrafiltration (5 times 10 ml) using a YM3 (3000 daltons cutoff) and then freeze dried. The yield is 0.63 grams of white crystalline powder. Characterization: i. Molecular weight.
  • Pepstatin can be conjugated to amino-arabinogalactan (2% amine by weight polysaccharide) through a N-hydroxy succinimide ester [Furuno, K. , et.al. (1983) J. Biochem 93: 249].
  • Arabinogalactan with a primary amine is prepared according to Example 2 (arabinogalactan-hydrazide) or example 5 or 7 (polylysine-arabinogalactan) .
  • Dissolve pepstatin A 250 mg
  • 1 ml of dimethylformamide Then add 50 mg l-ethyl-3 (3-dimethyl-aminopropyl)carbodiimide and 30 mg of N-hydroxy succimide.
  • Example 20 Arabinogalactan-WR2721 from thiophosphorylated arabinogalactan Thiophosphorylation of arabinogalactan.
  • Ten grams of anhydrous arabinogalactan is suspended in 50 ml of triethylphosphate. After the addition of 10.5 ml (75 millimole) of anhydrous triethyl amine, the suspension is cooled in an ice-water bath. To the cooled suspension is added dropwise with stirring 2.55 ml (25 millimole) of thiophosphoryl chloride. Once the addition is complete, the reaction mixture is warmed to room temperature and stirred for 72 hours.
  • the arabinogalactanyl thiophosphorodichloridate product is hydrolyzed by adding 50 ml of deionized ice-water and stirring for two hours.
  • the solvent, triethyl phosphate is removed from the reaction mixture by extraction with 2 times with 25 ml portions of chloroform.
  • the pH of the aqueous phase is adjusted to between 9 and 9.5 by the addition of 1 N sodium hydroxide.
  • the product is purified by ultra-filtration (50 ml to 10 ml, four cycles) using an Amicon YM3 (3000 dalton cutoff) ultrafiltration membrane. The final retentate is lyophilized to dryness.

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Abstract

L'invention se rapporte à un vecteur pouvant former un complexe avec un agent thérapeutique afin d'apporter celui-ci vers un récepteur cellulaire situé sur la surface d'un tissu cible, ce vecteur comprenant de l'arabinogalactane modifié au niveau d'un site par un reste fonctionnel afin de produire un dérivé d'une manière permettant de préserver l'affinité utile du dérivé envers le récepteur cellulaire. L'invention se rapporte également à des complexes ainsi formés.
PCT/US1992/005091 1992-06-17 1992-06-17 Derives d'arabinogalactane et leurs utilisations WO1993025239A1 (fr)

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CA002135295A CA2135295A1 (fr) 1992-06-17 1992-06-17 Derives de l'arabinogalactane, et leur utilisation
PCT/US1992/005091 WO1993025239A1 (fr) 1992-06-17 1992-06-17 Derives d'arabinogalactane et leurs utilisations
EP92914217A EP0646018A1 (fr) 1992-06-17 1992-06-17 Derives d'arabinogalactane et leurs utilisations
JP6501422A JPH07507794A (ja) 1992-06-17 1992-06-17 アラビノガラクタン誘導体及びその使用
NO944838A NO944838L (no) 1992-06-17 1994-12-14 Arabinogalaktanderivater og anvendelser derav

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Cited By (20)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994022484A1 (fr) * 1993-04-05 1994-10-13 Advanced Magnetics, Inc. Distribution selective de radioprotecteurs utilisant une molecule porteuse specifique d'un recepteur
FR2716625A1 (fr) * 1994-02-25 1995-09-01 Gouchet Franck Arno Préparation et utilisation de formes pharmaceutiques et cosmétiques contenant des composés d'inclusion avec les cyclodextrines de cystéamine, sels de cystéamine, ou dérivés de la cystéamine, sous forme de sel ou non.
WO1995019433A3 (fr) * 1994-01-12 1995-10-19 Targetech Inc Polynucleotides et oligonucleotides anti-hepatite b
WO1995034325A1 (fr) * 1994-06-16 1995-12-21 Advanced Magnetics, Inc. Apport d'agents therapeutiques a des recepteurs a l'aide de polysaccharides
US5567685A (en) * 1994-08-16 1996-10-22 Yissum Research Development Company Of The Hebrew University Of Jerusalem Water-Soluble polyene conjugate
WO1998022512A1 (fr) * 1996-11-20 1998-05-28 The University Of Montana Arabinogalactane soluble a l'eau comprenant des lipides
US5897987A (en) * 1996-03-25 1999-04-27 Advanced Reproduction Technologies, Inc. Use of arabinogalactan in cell cryopreservation media
US6011008A (en) * 1997-01-08 2000-01-04 Yissum Research Developement Company Of The Hebrew University Of Jerusalem Conjugates of biologically active substances
WO2001007486A1 (fr) * 1999-07-23 2001-02-01 Polygene Ltd. Composition polycationique biodegradable permettant d'administrer une macromolecule anionique
WO2001089579A3 (fr) * 2000-05-19 2002-08-29 Univ Minnesota Composition pour l'introduction de composes dans des cellules
US6929936B1 (en) * 1997-07-18 2005-08-16 Danisco A/S Composition comprising an enzyme having galactose oxidase activity and use thereof
US7148203B2 (en) 1997-03-11 2006-12-12 Regents Of The University Of Minnesota Nucleic acid transfer vector for the introduction of nucleic acid into the DNA of a cell
US7160682B2 (en) 1998-11-13 2007-01-09 Regents Of The University Of Minnesota Nucleic acid transfer vector for the introduction of nucleic acid into the DNA of a cell
WO2007039448A3 (fr) * 2005-09-20 2007-05-31 Uni Di Bologna Dipartimento Di Utilisation de conjugues de doxorubicine et d'albumine lactosaminee
WO2008123799A1 (fr) * 2007-04-04 2008-10-16 Institut Neftekhimii I Kataliza Rossiiskoi Akademii Nauk Arabinogalactanes modifiées et procédé de production associé
US7528234B2 (en) 2004-05-07 2009-05-05 Universita' Di Bologna Process for the preparation of doxorubicin conjugates with lactosaminated human albumin
CN102399238A (zh) * 2011-12-21 2012-04-04 开封明仁药业有限公司 氨磷汀的制备方法
US8227432B2 (en) 2002-04-22 2012-07-24 Regents Of The University Of Minnesota Transposon system and methods of use
RU2521649C1 (ru) * 2013-03-04 2014-07-10 Федеральное Государственное Бюджетное Учреждение Науки Институт Химии И Химической Технологии Сибирского Отделения Российской Академии Наук (Иххт Со Ран) Способ получения сульфатированных производных арабиногалактана
WO2014176389A1 (fr) * 2013-04-24 2014-10-30 Temple University - Of The Commonwealth System Of Higher Education Forme pharmaceutique solide contenant de l'arabinogalactane

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2330318A1 (fr) * 1998-04-27 1999-11-04 Larex, Inc. Derives de l'arabinogalactane et compositions les contenant

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE3200766A1 (de) * 1981-01-14 1982-09-16 Toyo Jozo Kk Praeparat mit ausgezeichneter absorptionseigenschaft
EP0331821A1 (fr) * 1988-02-03 1989-09-13 Kureha Kagaku Kogyo Kabushiki Kaisha Agent anti-viral
WO1990001295A1 (fr) * 1988-08-04 1990-02-22 Advanced Magnetics, Incorporated Agents de contraste d'irm de type endocytose induits par recepteur

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE3200766A1 (de) * 1981-01-14 1982-09-16 Toyo Jozo Kk Praeparat mit ausgezeichneter absorptionseigenschaft
EP0331821A1 (fr) * 1988-02-03 1989-09-13 Kureha Kagaku Kogyo Kabushiki Kaisha Agent anti-viral
WO1990001295A1 (fr) * 1988-08-04 1990-02-22 Advanced Magnetics, Incorporated Agents de contraste d'irm de type endocytose induits par recepteur

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
DATABASE WPIL Week 8550, Derwent Publications Ltd., London, GB; AN 85-314078 *

Cited By (26)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994022484A1 (fr) * 1993-04-05 1994-10-13 Advanced Magnetics, Inc. Distribution selective de radioprotecteurs utilisant une molecule porteuse specifique d'un recepteur
WO1995019433A3 (fr) * 1994-01-12 1995-10-19 Targetech Inc Polynucleotides et oligonucleotides anti-hepatite b
FR2716625A1 (fr) * 1994-02-25 1995-09-01 Gouchet Franck Arno Préparation et utilisation de formes pharmaceutiques et cosmétiques contenant des composés d'inclusion avec les cyclodextrines de cystéamine, sels de cystéamine, ou dérivés de la cystéamine, sous forme de sel ou non.
WO1995034325A1 (fr) * 1994-06-16 1995-12-21 Advanced Magnetics, Inc. Apport d'agents therapeutiques a des recepteurs a l'aide de polysaccharides
US5567685A (en) * 1994-08-16 1996-10-22 Yissum Research Development Company Of The Hebrew University Of Jerusalem Water-Soluble polyene conjugate
US6593138B1 (en) 1996-03-25 2003-07-15 Bio-Origyn Llc Cryopreserving somatic cells with an arabinogalactan containing cryopreservation medium
US5897987A (en) * 1996-03-25 1999-04-27 Advanced Reproduction Technologies, Inc. Use of arabinogalactan in cell cryopreservation media
US6258796B1 (en) 1996-11-20 2001-07-10 The University Of Montana Water soluble lipidated arabinogalactan
US6303584B1 (en) 1996-11-20 2001-10-16 The University Of Montana Water soluble lipidated arabinogalactan
WO1998022512A1 (fr) * 1996-11-20 1998-05-28 The University Of Montana Arabinogalactane soluble a l'eau comprenant des lipides
US6011008A (en) * 1997-01-08 2000-01-04 Yissum Research Developement Company Of The Hebrew University Of Jerusalem Conjugates of biologically active substances
US7148203B2 (en) 1997-03-11 2006-12-12 Regents Of The University Of Minnesota Nucleic acid transfer vector for the introduction of nucleic acid into the DNA of a cell
US6929936B1 (en) * 1997-07-18 2005-08-16 Danisco A/S Composition comprising an enzyme having galactose oxidase activity and use thereof
US7160682B2 (en) 1998-11-13 2007-01-09 Regents Of The University Of Minnesota Nucleic acid transfer vector for the introduction of nucleic acid into the DNA of a cell
WO2001007486A1 (fr) * 1999-07-23 2001-02-01 Polygene Ltd. Composition polycationique biodegradable permettant d'administrer une macromolecule anionique
US7001891B1 (en) * 1999-07-23 2006-02-21 Efrat Biopolymers Limited Biodegradable polycation composition for delivery of an anionic macromolecule
WO2001089579A3 (fr) * 2000-05-19 2002-08-29 Univ Minnesota Composition pour l'introduction de composes dans des cellules
US8227432B2 (en) 2002-04-22 2012-07-24 Regents Of The University Of Minnesota Transposon system and methods of use
US7528234B2 (en) 2004-05-07 2009-05-05 Universita' Di Bologna Process for the preparation of doxorubicin conjugates with lactosaminated human albumin
WO2007039448A3 (fr) * 2005-09-20 2007-05-31 Uni Di Bologna Dipartimento Di Utilisation de conjugues de doxorubicine et d'albumine lactosaminee
WO2008123799A1 (fr) * 2007-04-04 2008-10-16 Institut Neftekhimii I Kataliza Rossiiskoi Akademii Nauk Arabinogalactanes modifiées et procédé de production associé
RU2359975C2 (ru) * 2007-04-04 2009-06-27 Институт нефтехимии и катализа РАН Способ получения модифицированных арабиногалактанов
CN102399238A (zh) * 2011-12-21 2012-04-04 开封明仁药业有限公司 氨磷汀的制备方法
RU2521649C1 (ru) * 2013-03-04 2014-07-10 Федеральное Государственное Бюджетное Учреждение Науки Институт Химии И Химической Технологии Сибирского Отделения Российской Академии Наук (Иххт Со Ран) Способ получения сульфатированных производных арабиногалактана
WO2014176389A1 (fr) * 2013-04-24 2014-10-30 Temple University - Of The Commonwealth System Of Higher Education Forme pharmaceutique solide contenant de l'arabinogalactane
US10994013B2 (en) 2013-04-24 2021-05-04 Temple University—Of the Commonwealth System of Higher Education Solid dosage form containing arabinogalactan

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EP0646018A1 (fr) 1995-04-05
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JPH07507794A (ja) 1995-08-31

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