CA2135295A1 - Arabinogalactan derivatives and uses thereof - Google Patents
Arabinogalactan derivatives and uses thereofInfo
- Publication number
- CA2135295A1 CA2135295A1 CA002135295A CA2135295A CA2135295A1 CA 2135295 A1 CA2135295 A1 CA 2135295A1 CA 002135295 A CA002135295 A CA 002135295A CA 2135295 A CA2135295 A CA 2135295A CA 2135295 A1 CA2135295 A1 CA 2135295A1
- Authority
- CA
- Canada
- Prior art keywords
- arabinogalactan
- complex
- agent
- cell receptor
- therapeutic agent
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
Classifications
-
- 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
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Materials Engineering (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Polymers & Plastics (AREA)
- Organic Chemistry (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Polysaccharides And Polysaccharide Derivatives (AREA)
Abstract
There are provided a carrier for forming a complex with a therapeutic agent for delivery thereof to a cell receptor located on the surface of a target tissue comprising: arabinogalactan modified at a site by a functional residue to produce a derivative in a manner that preserves the useful affinity of the derivative for the cell receptor, and complexes thereof.
Description
W093/2~239 PCT/US92/05091 21~5295 ARABINOGAL~CTAN DERIVATIVES AND USES THEREOF
S Technical Field This invention relates to the synthesis and methods of use of therapeutic agents targeted to cells, especially hepatocytes.
Backaround Art The safety and efficacy of a therapeutic agent is a function of (i) its intrinsic biological acti~ity and ~ii) the biodistribution achieved after its administration. Many potentially useful therapeutic agents possess a biochemical activity ameliorating a par~icular pathological condition, lS 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 u e of therapeutic agents with established antiviral activity, or 20 agents with established anti-cancer activity. There is a need for new compounds to target therapeutic agents to the specific cells that are the source of some pathological condition, and to reduce the concentration attained in unaffected, normal tissues. Targeting is the modification 25 of a therapeutic agent so that after injection or oral administration the uptake by a specific population of cells is increased re1ative to uptake of the unmodified agent. By targeting compounds with established and beneficial biological activity to specific tissues, compounds whose use is currently limited by side effects might become safe and , efficacious drugs. 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, 35 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 W093~2~239 2 1 3 5 ~ 3 ~ PCT/US92/0509l 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 5 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 10 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 15 residues to non-glycoproteins such as human albumin.
When attaching diagnostic and therapeutic agents to a receptor-recognizing carrier molecule, targeting can be achieved only if the affinity of the carrier for the receptor is maintained. The differential reactivity of the 20 protein amine and carbohydrate hydroxyl groups of glycoprotein carriers, e.g. asialofetuin, i5 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 25 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. Wu, Marcel Dekker Inc. pp. 235-264. In contrast, a 30 polysaccharide such as arabinogalactan offers no polypeptide ~ amino groups distal from the receptor binding site that can ; be modified for the purposes of retaining the asialoglucoprotein receptor binding activity. In spite of the obvious strategy for modification of glycoproteins with 35 retention of receptor binding activity, their use for targeted, parenteral pharmaceuticals is subject to several problems.
.
(i) Glycoproteins are prepared from animal cells and insuring noncontamination with human infective viral pathogens is a major issue.
(ii) Glycoproteins will not generally tolerate organic 5 solvents during conjugate synthesis, because such solvents frequently lead to a loss of biological activity and denaturation.
(iii) Glycoproteins can be toxic and/or antigenic.
(iv) Glycoproteins in their native form, e.g. fetuin, 10 do not afford galactose resides and must be desialylated to produce a carrier which interacts with the receptor.
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 15 arabinogalactan is the American Western larch (Larix occidentalis). Arabinogalactan from this source is used as a binder, emulsifier or stabilizer in foods. It consists ~f a largely 1-3 linked D-galactose bac~bone with 1-6 linked branch chains of L-arabinoses and D-galactoses at 20 practically every residue on the backbone. In larch arabinogalactans the ratio of galactose to arabinose is between 5 to 1 and 10 to l, while arabinogalactans from plant sources in general range from about 1 to 4 to about 10 to l [Clarke, A.E., Anderson, R.L., Stone, B.A.;
25 Phytochemistry (1979) 18: 521-40~. Like many polysaccharides, arabinogalactans have different molecular weights with values of about 1-2 million to a~out 10,000 daltons ~Blake, J.D., Clarke, M.L., Jansson, P.E., Carbohydr Res tl9833 115: 265-272] having been reported. It has been 30 shown that L-arabinose and D-galactose interact with the asialoglycoprotein receptor while common monosaccharides like glucose or mannose do not [Lee, Haekyung, Kelm, Sorge, Teruo, Yoshino, Schauer, Roland Biol. Chem., Hoppe-Seyler (1988) 369, 705-714].
Some derivatives of arabinogalactan have been previously prepared. Graft copolymers have been used in paper manufacturing [SUl285094] and in soil treatments WO 93f25239 2 1 3 5 ~ .S PCI/US92/0509~
[JP1051198]. Arabinogalactan sulfate has been used to form salts with drugs ~o influence drug absorption and prolong drug action [US4609640]. Acidic forms of arabinogalactan occur naturally having a composition which includes uranic 5 acid ~Clarke, A.E., Anderson, R.L., Stone, B.A., Phytochemistry (1979) 18, 521-40], and have also been prepared from arabinogalactan [JP60219202]. Derivatives of arabinogalactan with substituent alkyl, allyl cyano, halo or amino groups, and conjugates with organic acids and enzyme 10 protein have been disclosed, wherein the carbohydrate is used as a carrler, adsorbent or resin ~JP60219201]. In some cases arabinogalactan has been highly derivatized in a manner likely to destroy its interaction with the asialoglycoprotein receptor. For example, in some cases as 15 many as 50% of the hydroxyl groups of arabinogalactan have been modified ~JP60219201], but the affinity, or lack thereof, of arabinogalactan derivatives for the asialoglycoprotein receptor has not been studied.
Summary of Invention The present invention provides for derivatives of arabinogalactan which can be used to target therapeutic - agents to the cells possessing the asialoglycoprotein receptor.
~- The use of the polysaccharide arabinogalactan to target 25 therapeutic agents to cells via the asialoglycoprotein receptor, a feature of the current invention, overcomes problems encountered when glycoproteins are used for this , purpose.
(i) A polysaccharide like arabinogalactan originating 30 from a plant source is unlikely to be contaminated with human viral pathogens.
(ii) Since arabinogalactan is a polysaccharide, it will tolerate exposure to organic solvents, which normally denature proteins during conjugate synthesis. Composed 35 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 W093/25239 2 1 3 5 2 ~ 5 P~T/US92~05091 ' unwanted synthetic byproducts. This advantage is evident in the examples below.
(iii) Arabinogalactan has low toxicity and antigenicity.
(iv) 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.
We have discovered that 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 15 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.
In some instances targeting may be employed to deliver 20 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 25 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. The use of the term "therapeutic agent" in this description and the accompanying claims, includes agents which are protective from toxic chemicals or radiation.
The arabinogalactan derivatives of the invention must interact strongly with the asialoglycoprotein receptor, so 35 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 i~
W093/2~239 ~ PCT/US92/0509l 2~ 3~29`-~ 6 -with the receptor is presented.
In one embodiment the antiviral therapeutic agent adenosine arabinoside mono-5'-phosphate (ARA-AMP) is coupled to arabinogalactan. In addition, ARA-A or acyclovir, both 5 antiviral therapeutic agents, may also separately be coupled to arabino-galactans. In another embodiment the radioprotective agent S-2-(3 aminopropylamino) ethyl-thiophosphoric acid (known as WR2721) is attached to arabinogalactan. The invention provides methods and 10 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.
Detailed Descri~tion of Specific Embodiments The 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 25 to arabinose of 5 to 1 as determined by the alditol acetate method. It binds the asialoglycoprotein receptor on h~patocytes tJosephson Groman et al. Mag. Res. Imag. (1990) 8: 637-646]. It has been shown that L-arabinose, and D-galactose interact with the asialoglycoprotein receptor 30 while, for example, common monosaccharides like glucose or mannose do not [Lee, Haekyung, Kelm, et al., Biol. Chem., ~oppe-Seyler (1988) 369: 705-714]. It has also been shown that an underivatized 4-hydroxy group on galactose and the clustering of suitable sugars, as is displayed by highly 35 branched polysaccharides like arabinogalactan, are important factors in binding the asialoglycoprotein receptor. Given these requirements, and based on the above composition and W093/25239 2 1 ~ ~ 2 ~ 5 PCT/US92/oSOgl structure, arabinogalactan is distinguishable from other polysaccharides including dextrans, starches, celluloses, inulins, 1-4 linked galactan and gum arabi Though chemically distinguishable from arabinogalactan, gum arabic 5 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 10 of arabinogalactan which interact with the asialoglycoprotein receptor. When an ara~inogalactan derivative is recognized by the asialoglycoprotein receptor, a therapeutic agent zan be targeted into the cells possessing that receptor, chiefly the hepatocytes.
15 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, Groman et al., Mag. Res. Imag. (1990) 8:637-646]. Hepatocytes are the 20 predominant cell possessing this receptor, and endocytose a large proportion of injected radiolabelled asialoglycoproteins [Hub~ard, Wilson et. al., J. Cell Biol.
(1979) 83:47-64]. However, asialoglycoprotein receptors have been detected on Kupffer cells ~Lee, Haekyung, et al.
25 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]. Useful amounts of a therapeutic agent may be targeted to any asialoglycoprotein 30 receptor positive cell. Similarly 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 35 use is associated with serious neurological side effects ~Lok, A.S., Wilson, L.A. et al., J. Antimicrob. Chemother.
(1984) 14: 93-9; Hoffnagel, J.H. et al., J. Hepatol. (1986) W093/25239 ` PCT~US92~05091 21~5~
S Technical Field This invention relates to the synthesis and methods of use of therapeutic agents targeted to cells, especially hepatocytes.
Backaround Art The safety and efficacy of a therapeutic agent is a function of (i) its intrinsic biological acti~ity and ~ii) the biodistribution achieved after its administration. Many potentially useful therapeutic agents possess a biochemical activity ameliorating a par~icular pathological condition, lS 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 u e of therapeutic agents with established antiviral activity, or 20 agents with established anti-cancer activity. There is a need for new compounds to target therapeutic agents to the specific cells that are the source of some pathological condition, and to reduce the concentration attained in unaffected, normal tissues. Targeting is the modification 25 of a therapeutic agent so that after injection or oral administration the uptake by a specific population of cells is increased re1ative to uptake of the unmodified agent. By targeting compounds with established and beneficial biological activity to specific tissues, compounds whose use is currently limited by side effects might become safe and , efficacious drugs. 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, 35 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 W093~2~239 2 1 3 5 ~ 3 ~ PCT/US92/0509l 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 5 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 10 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 15 residues to non-glycoproteins such as human albumin.
When attaching diagnostic and therapeutic agents to a receptor-recognizing carrier molecule, targeting can be achieved only if the affinity of the carrier for the receptor is maintained. The differential reactivity of the 20 protein amine and carbohydrate hydroxyl groups of glycoprotein carriers, e.g. asialofetuin, i5 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 25 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. Wu, Marcel Dekker Inc. pp. 235-264. In contrast, a 30 polysaccharide such as arabinogalactan offers no polypeptide ~ amino groups distal from the receptor binding site that can ; be modified for the purposes of retaining the asialoglucoprotein receptor binding activity. In spite of the obvious strategy for modification of glycoproteins with 35 retention of receptor binding activity, their use for targeted, parenteral pharmaceuticals is subject to several problems.
.
(i) Glycoproteins are prepared from animal cells and insuring noncontamination with human infective viral pathogens is a major issue.
(ii) Glycoproteins will not generally tolerate organic 5 solvents during conjugate synthesis, because such solvents frequently lead to a loss of biological activity and denaturation.
(iii) Glycoproteins can be toxic and/or antigenic.
(iv) Glycoproteins in their native form, e.g. fetuin, 10 do not afford galactose resides and must be desialylated to produce a carrier which interacts with the receptor.
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 15 arabinogalactan is the American Western larch (Larix occidentalis). Arabinogalactan from this source is used as a binder, emulsifier or stabilizer in foods. It consists ~f a largely 1-3 linked D-galactose bac~bone with 1-6 linked branch chains of L-arabinoses and D-galactoses at 20 practically every residue on the backbone. In larch arabinogalactans the ratio of galactose to arabinose is between 5 to 1 and 10 to l, while arabinogalactans from plant sources in general range from about 1 to 4 to about 10 to l [Clarke, A.E., Anderson, R.L., Stone, B.A.;
25 Phytochemistry (1979) 18: 521-40~. Like many polysaccharides, arabinogalactans have different molecular weights with values of about 1-2 million to a~out 10,000 daltons ~Blake, J.D., Clarke, M.L., Jansson, P.E., Carbohydr Res tl9833 115: 265-272] having been reported. It has been 30 shown that L-arabinose and D-galactose interact with the asialoglycoprotein receptor while common monosaccharides like glucose or mannose do not [Lee, Haekyung, Kelm, Sorge, Teruo, Yoshino, Schauer, Roland Biol. Chem., Hoppe-Seyler (1988) 369, 705-714].
Some derivatives of arabinogalactan have been previously prepared. Graft copolymers have been used in paper manufacturing [SUl285094] and in soil treatments WO 93f25239 2 1 3 5 ~ .S PCI/US92/0509~
[JP1051198]. Arabinogalactan sulfate has been used to form salts with drugs ~o influence drug absorption and prolong drug action [US4609640]. Acidic forms of arabinogalactan occur naturally having a composition which includes uranic 5 acid ~Clarke, A.E., Anderson, R.L., Stone, B.A., Phytochemistry (1979) 18, 521-40], and have also been prepared from arabinogalactan [JP60219202]. Derivatives of arabinogalactan with substituent alkyl, allyl cyano, halo or amino groups, and conjugates with organic acids and enzyme 10 protein have been disclosed, wherein the carbohydrate is used as a carrler, adsorbent or resin ~JP60219201]. In some cases arabinogalactan has been highly derivatized in a manner likely to destroy its interaction with the asialoglycoprotein receptor. For example, in some cases as 15 many as 50% of the hydroxyl groups of arabinogalactan have been modified ~JP60219201], but the affinity, or lack thereof, of arabinogalactan derivatives for the asialoglycoprotein receptor has not been studied.
Summary of Invention The present invention provides for derivatives of arabinogalactan which can be used to target therapeutic - agents to the cells possessing the asialoglycoprotein receptor.
~- The use of the polysaccharide arabinogalactan to target 25 therapeutic agents to cells via the asialoglycoprotein receptor, a feature of the current invention, overcomes problems encountered when glycoproteins are used for this , purpose.
(i) A polysaccharide like arabinogalactan originating 30 from a plant source is unlikely to be contaminated with human viral pathogens.
(ii) Since arabinogalactan is a polysaccharide, it will tolerate exposure to organic solvents, which normally denature proteins during conjugate synthesis. Composed 35 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 W093/25239 2 1 3 5 2 ~ 5 P~T/US92~05091 ' unwanted synthetic byproducts. This advantage is evident in the examples below.
(iii) Arabinogalactan has low toxicity and antigenicity.
(iv) 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.
We have discovered that 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 15 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.
In some instances targeting may be employed to deliver 20 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 25 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. The use of the term "therapeutic agent" in this description and the accompanying claims, includes agents which are protective from toxic chemicals or radiation.
The arabinogalactan derivatives of the invention must interact strongly with the asialoglycoprotein receptor, so 35 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 i~
W093/2~239 ~ PCT/US92/0509l 2~ 3~29`-~ 6 -with the receptor is presented.
In one embodiment the antiviral therapeutic agent adenosine arabinoside mono-5'-phosphate (ARA-AMP) is coupled to arabinogalactan. In addition, ARA-A or acyclovir, both 5 antiviral therapeutic agents, may also separately be coupled to arabino-galactans. In another embodiment the radioprotective agent S-2-(3 aminopropylamino) ethyl-thiophosphoric acid (known as WR2721) is attached to arabinogalactan. The invention provides methods and 10 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.
Detailed Descri~tion of Specific Embodiments The 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 25 to arabinose of 5 to 1 as determined by the alditol acetate method. It binds the asialoglycoprotein receptor on h~patocytes tJosephson Groman et al. Mag. Res. Imag. (1990) 8: 637-646]. It has been shown that L-arabinose, and D-galactose interact with the asialoglycoprotein receptor 30 while, for example, common monosaccharides like glucose or mannose do not [Lee, Haekyung, Kelm, et al., Biol. Chem., ~oppe-Seyler (1988) 369: 705-714]. It has also been shown that an underivatized 4-hydroxy group on galactose and the clustering of suitable sugars, as is displayed by highly 35 branched polysaccharides like arabinogalactan, are important factors in binding the asialoglycoprotein receptor. Given these requirements, and based on the above composition and W093/25239 2 1 ~ ~ 2 ~ 5 PCT/US92/oSOgl structure, arabinogalactan is distinguishable from other polysaccharides including dextrans, starches, celluloses, inulins, 1-4 linked galactan and gum arabi Though chemically distinguishable from arabinogalactan, gum arabic 5 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 10 of arabinogalactan which interact with the asialoglycoprotein receptor. When an ara~inogalactan derivative is recognized by the asialoglycoprotein receptor, a therapeutic agent zan be targeted into the cells possessing that receptor, chiefly the hepatocytes.
15 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, Groman et al., Mag. Res. Imag. (1990) 8:637-646]. Hepatocytes are the 20 predominant cell possessing this receptor, and endocytose a large proportion of injected radiolabelled asialoglycoproteins [Hub~ard, Wilson et. al., J. Cell Biol.
(1979) 83:47-64]. However, asialoglycoprotein receptors have been detected on Kupffer cells ~Lee, Haekyung, et al.
25 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]. Useful amounts of a therapeutic agent may be targeted to any asialoglycoprotein 30 receptor positive cell. Similarly 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 35 use is associated with serious neurological side effects ~Lok, A.S., Wilson, L.A. et al., J. Antimicrob. Chemother.
(1984) 14: 93-9; Hoffnagel, J.H. et al., J. Hepatol. (1986) W093/25239 ` PCT~US92~05091 21~5~
3: S73-80]. ARA-AMP conjugated to arabinogalactan, and targeted to asialoglycoprotein receptor possessing cells where viral replication is ongoing thepatocytes), is expected to reduce unwanted side effects by reducing the 5 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 10 be used for this purpose include acyclovir and Ara-A~
A second type of anti-viral agent that can be targeted with the teachings of the invention are antibodies. In this context antibodies may include polyclonal antibodies, monoclonal antibodies or antibody fragments. The natural 15 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 (l991), p. 28]. In particular hepatitis B virus replication 20 occurs within the hepatocytes of the liver and antibodies to viral antigens cannot directly bind the virus during this replication. If an antibody to a hepatitis B viral protein is conjugated to arabinogalactan, it will be targeted via the asialoglycoprotein receptor to the cytoplasm of 25 hepatocytes. Within the cytoplasm the antiviral antibody will bind replicating hepatitis B virus and become an effective therapeutic agent.
Some of the arabinogalactan derivatives described have ~, no known pharmacological activity, other than their ability 30 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 35 receptor, principally the hepatocytes of the liver. The carboxyl groups afforded by the succinyl-arabinogalactan, glutaryl-arabinogalactan and DTPA-arabinogalactan conjugates W093/2~239 213 S 2 n ~ PCT/US92/05091 (Examples 10-12) can be used to attach molecules through the use of carboniimides or other agents. The amino groups afforded by the arabinogalactan hydrazide (Example 3) or poly-L-lysine arabinogalactan (Examples 6, 8) can also be 5 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., Leonetti, J.P., Gagnor, C., Lemaitre, M., Lebleu, B., Nucleic Acids Res (1989) 17: 9341-15 50]. In addition to poly-L-Lysine, other polymeric molecules, such as dextrin, dextran, or albumin may be coupled to arabinogalactan.
In another embodiment, galactose oxidase treatment of arabinogalactan can be used to create aldehyde groups. The 20 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.
Similarly 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., He, S., Zhang, Z., 7th International Conference on Chemical 30 Modifiers of Cancer Treatment (1991), Clearwater, FA 338-340] or chemotherapy [Schein, P.S, International Conference on Chemical Modifiers of Cancer Treatment (1991), -Clearwater, FA 341-342]. The utility of WR2721 as a chemoprotectant has been objected to based on the lack of 35 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 W093/25239 PCTtUS92/05091 ~13 .~ 2 ~ ~
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 5 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, 10 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], 15 cationic aminothiols generally, glutathiols, and vitamin E
derivatives.
After synthesis, the interaction of the arabinogalactan derivative with the asialoglycoprotein receptor can be determined in vivo. The ability of a derivative to interact 20 with the asialoglycoprotein receptor is assessed by its ability to block the clearance of a substance recognized to ~ interact with the asiàloglycoprotein receptor based on --~ earlier work. An arabinogalactan coated superparamagnetic ~ iron oxide colloid interacts with this receptor and a ~ ~ .
25 quantitative assay for its clearance has been described below. In the absence of a blocking agent, 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 30 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.
To obtain the blood half-life a Sprague-Dawley rat (200-300 grams) is anesthetized (lO0 mg/kg of Inactin) and injected with a defined dose of a blocking agent, followed by an arabinogalactan coated superparamagnetic iron oxide at W093t25239 - 2 1 3 5 ~ 9 - PCT/USg2/05091 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/Tl the 5 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 10 a blocking agent (receptor binding activity). With antibodies and enzymes, covalent modification especially high levels of covalent modification, generally decreases or - destroys biological function. Thus it i~ surprising that arabinogalactan tolerates random modification with excellent 15 retention of its receptor-recognizing biological activity.
In fact 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 20 unknown. In contrast, 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 25 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.
If an arabinogalactan conjugate is inactive in the 30 blocking assay, i.e., does not prolong blood half-life, conditions used in conjugate synthesis can be adjusted to achieve a lower degree of modification. Alternatively, the $
modification strategy employed may be dropped altogether and a different procedure employed.
W093/25239 PC~/US92/05091 ~1352~
Interactions of arabinogalactan deri~atives with the asialoglycoprotei~ receptor Agent Dose Blocking Agent Half-life (mg/kg) (min) , _ _ None none 2.8 . _ _.
lactose 300 8.0 . ,. ~
arabinogalactan 150 33.2 phosphoryl- 150 51.0 arabinogalactan .
(Example 9) . .
succinyl 150 213 arabinogalactan (Example 11) _ arabinogalactan-AMP 15Q ~100 _ (Example 4) I
arabinogalactan- 150 86 WR2721 (Example 15) I _ arabinogalactan 150 7.3 acetate (Example 18) . .
arabinogalactan 150 40.8 propionate (Example 19) _ _ . ,.
The examples below demonstrate that arabinogalactan can be modified by the addition of phosphoryl, sulfhydryl, amino, carboxyl, halo, or acylimidazol groups, with receptor 30 binding activity being unaffected. The initial modification is performed on the hydroxyl groups on the arabinogalactan.
~- W093/25239 21~ S 2 ~J - PCT/US92/05091 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 5 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 10 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, l991).
Reagents used to couple proteins to solid phase amino or 15 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 àrabinogalactan to provide useful pharmaceutical agents are listed in Table 2.
_ r W093~2~239 ~ PCT/US92/05091 ,~
Therapeutic agents that have been or might be attached to ara~inogalactan or arabinogalactan derivatives , _ _ _ _ ~
Agent Class Reference I . . ,_ Ara-AMP antiviral activity Example 5 _ _ _ WR2721 chemo- and Example 14 radioprotective activity .
Pepstatin anti-inflammatory Example 17 activity : lO gene therapy Examples 6, 8 DNA .
,~ _ .
Antisense antiviral activity Examples 6, 8 nucleic acid : Antibody to antiviral activity : 15 hepatitis .~
. ~ Steriod ~: anti-inflammatory activity I _ _ _ _ Superoxide dismutase anti-inflammatory ¦ activity _ t Examples 2S '~
Ex~mple 1: Bromination of arabinogalactan.
The arabinogalactan (AG) used is from the Western Larch and chromatographs produre a single peak of about 20,000 daltons by size exclusion chromatography.
Ten grams of arabinogalactan are dissolved in 35 ml of - W093/25239 ~1 3 5 2 ~ ~ PCT/US92/0~091 a 7.1% (w/v) solution of Zn(BF4)2. Fifty ml of epibromohydrin is added and the solution stirred for so minutes at 100C. The bromo-arabinogalactan is precipitated in 150 ml cold (4-C) acetone, redissolved in water, and 5 precipitated in 150 ml cold ethanol. Analysis of the product for bromine showed 0.7 milliequivalents bromide per gram product.
Example 2: Treatment of arabinogala~tan with sodium borohydr~e.
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 NaBH4.
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 20 arabinogalactan produced no dye, indicating essentially complete reduction.
Example 3: ~ydrazino-arabinogalactan.
. .
Ten grams of reduced arabinogalactan (Example 2~ is dissolved in 35 ml of a 7.1% (w/v) aqueous solution of 25 Zn(BF~)2. Fifty ml of epibromohydrin is added and the solution stirred for 90 minutes at lOO 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 30 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. The hydrazido-arabinogalactan ^~
is precipitated in 150 ml cold (4-C) acetone, redissolved in water and precipitated in 150 ml cold ethanol. The product 35 hydrazide content is analyzed by acid-base titration and ~howed 0~25 milliequivalents hydrazide per gram of product.
W O 93/25239 PC~r/US92/05091 t -21352~5 Ex~mple 4: Arabino~alactan conjugated to adenosine 5~ monopho~phate ~AMP) One gram (2.9 mmoles) of adenosine 5'-monophosphate (AMP) is dissolved in 20 ml water with the addition of !-5 sodium bicarbonate powder. Arabinogalactan-hydrazide (0.6 g, example 2) is added and the pH adjusted to 7.5 with sodium hydroxide. One gram (5.2 mmoles) of 1-ethyl-3,4-- dimethylaminopropyl) carbodiimide is added and the reaction maintained at room temperature for 64 hours. The product is 10 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 15 buffer of 0.1 mM, pH 7.0 phosphate buffer at flow rate o.5 ml/min). A single broad pea~ 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 ~G-AMP product, based on the comparison of HPLC area under the 20 curve monitoring at 260 nm is 0.24, indicating approximately a 95% conversion of available hydrazide groups. The UV/VlS
, , 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 -~ 25 molecular weight approximately equivalent to underivatized arabinoga~lactan, about 20,000 daltons.
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 30 colloid, extending the half-life of the colloid to greater than 100 minutes (Table 1).
Ex~mple 5: Arabinogalactan conjugated to adenine 7 ar~binoside s~ monopho~phate (ARA-AMP).
One gram (2.9 mmoles) of adenine arabinoside 5'-35 monophosphate (ARA-AMP) is dissolved in 20 ml water with the addition of sodium bicarbonate powder. 0.6 grams of arabinogalactan-hydrazide (Example 2 above) is then added ' .
W093~25239 ~1 352~ - PCT/US92/0509l and the pH adjusted to 7.5 with the addition of sodium hydroxide. One gram (5.2 mg) of 1-ethyl-3,4-dimethylaminopropyl) carbodiimide is added and the reaction maintained at room temperature ~or 64 hours. The product is 5 purif~ed by ultrafiltration using an Amicon YM3 ultrafilter, and then further purified by precipitation fram ethanol and dried, yielding 280 mg of product. As with AG-AMP (Example 3), the strong cation exchange chromatography showed a single broad peak centered at 5.7 minutes and no measurable 10 residual unreacted ARA-AMP. The UV/VIS spectrum of AG-ARA-AMP was ~irtually identical to an ARA-AMP standard. Based _ on the area under the curve at an optical density of 260 nanometers and in comparison with an AMP standard, which is assumed to have the same extinction coefficient as ARA-AMP, 15 the product has 0.124 milli-equivalents of ARA-AMP per gram.
Ex~mple 6: Poly~L)lysyl-~rabinog~la~t~n ~prep~re~ by re~uctive ~mination).
Poly~L)lysine hydrochloride (1,000-4,000 daltons, 0.5 grams) is dissolved in 2 ml borate buffer (0.2M) and the pH
20 adjusted to 9.0 with sodium hydroxide. 100 mq arabinogalactan and 50 mg sodium cyanoborohydride is added.
~- and the reaction heated for 24 hours at 50-C. The product mixture is puriied using an Amicon YM3 ultrafilter. The retentate containing the polylysine-arabinogalactan 25 conjugate showed a positive ninhydrin test for amine and positive anthrone test for polysaccharide. The yield was 30 mg. Si~e exclusion high performance liquid chromatography (Amicon Cellufine GC200M) showed a product having a molecular weight approximately equal to the sum of molecular 30 weights of arabinogalactan and poly~L)lysine or about 25,000 daltons.
Ex~mple 7: Acylimidazole-arabinogalact~n.
Three grams of anhydrous arabinogalactan is suspended in 5 ml of anhydrous peroxide free dioxane. While stirring, 35 1.62 gm (lOmmole) of N,N'-carbonyl diimidazole, dissolved in 10 ml of dioxane, is added in a single portion. After stirring for 20 minutes the acylimidazol-arabinogalactan is W093/25~39 PCT/US92/05U91 ,~~
- ~8 -collected by fil~ration (medium frit). The praduct is washed with 25 ml of dioxane and refiltered. A second dioxane titration is performed. The product is next titrated with 25 ml of apf-diethyl ether and then vacuum 5 dried. Yield is 2.9 gm.
Ex~mple 8: Poly~L)ly~ine-arabi~ogalactan ~prepared rom acylimidazole-arabinogalactan).
One gram of acylimidazole-arabinogalactan (Example 7 and 0.2 grams poly~L)lysine (1,000-4,000 daltons) is 10 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 5C. The product is isolated first by precipitation in ethanol, and then purified using an Amicon YM10 ultrafilter. The retentate shows a positive 15 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.
The product poly(L)lysyl-arabinogalactan is analyzed by cation exchange chromatography HPLC (Rainin Synchropak 20 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.
The product, arabinogalactan-polylysine, elutes with a retention time of 3.9 minutes whereas unconjugated polylysine elutes with a retention time of 9.3 minutes.
25 Poly(L)lysine bound to arabinogalactan is verified by its UV
speatrum.
Example 9: Phosphoryl-arabinogalactan.
Two grams of arabinogalactan are dissolved in 20 ml formamide and 4 ml triethylamine. Ten grams polyphosphoric 30 acid are added and the reaction stirred for 1~ hours. The product is brought to pH 9 with 45~ NaOH and ultrafiltered in a SO ml stirred cell with a 3,000 molecular weight cutoff membrane ~Amicon), bringing the volume from 50 ml to 10 ml twice. The ultrafiltered product is precipitated into 500 35 ml cold acetone (4-C), redissolved, and precipitated in SOO
ml cold ethanol. The product showed 0.21 milli-equivalents of phosphate per gram of product both by acid base titration W093t25239 _ 2 1 3 5 2 ~ ;~ PCTiUS92/05091 -- lg --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).
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).
10 Example 10: Treatment of arabinogalactan with galactose oxida~e (GO).
- Ten grams of arabinogalactan is dissolved to a total volume of about 50 ml in O.1 M potassium phosphate buffer, pH = 6Ø To the resultant solution is added 225 units of 15 galactose oxidase dissolved in about 2 ml of the same buffer. The oxidation is allowed to proceed for 24 hours at room temperature. The H2O2 content is found to be about 3 mg/ml, as measured by peroxide test strips. A second addition of 22S units of GO is made to the reaction mixture.
20 After another 24 hour reaction period the peroxide content is found to be unchanged from the result of the first GO
treatment at about 3 mg/ml. Twenty milligrams of catalase ~--(dry solid) is added to decompose the peroxide. After standing at room temperature overnight the contents of the -- 25 flask are found to be free of peroxide. }
Product Purification. Ten grams of mixed bed resin, MB-l is added to the flask. After stirring for 30 minutes the solution is decanted into and passed through a short column containing an additional 5 grams of MB-l resin. The 30 pH neutral solution is found to be free of any protein amines by reaction with ninhydrin. The product is isolated by precipitation from 5-C cooled absolute ethanol. The precipitate is collected by filtration. The aldehyde content of this product is found to be between 3 and 5 times 35 greater than the aldehyde content of native arabinogalactan.
Yield is lO grams.
Determination of the Number of Aldehyde Groups: The 3---W093/25239 PCT/U~92/0509l ~
~13~
methyl-2-benzothiazolone hydrazone test for aldehyde was used to compare arabinogalactan starting material to poly-aldehydic arabinogalactan. Based on absorbance measured at 670 nm, this poly-aldehydic arabinogalactan has 0.34 milli-5 equivalents aldehyde per gram of ara~inogalactan.Ex~mple 11: ~uGcinyl-arabinogalactan.
Purified arabinogalactan (16.0 g, 0.70 mmol) and succinyl anhydride (10.0 g, 100 mmol) were dissolved in DMSO
(200 ml) at 60-C. After 1.0 hour, the clear, light yellow 10 solution was cooled to ambient temperature and allowed to stir for an additional 48 h. The DMSO solution was added to - H2O (200 ml), filtered on an Amicon YM3 ultrafiltration membrane and washed with H2O (3 times with 250 ml). The solution remaining on the membrane was frozen and 15 lyophilized. Yield of white powder: 20.~ g. IR (KBr):
1732 cm1 (C=O). Titration of an aqueous solution of the conjugate with G.01 N NaOH indicated the presence of 1.96 milli-equivalents succinate per gram of succinyl-arabinogalactan.
The activity of succinyl-arabinogalactan is 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 213 minutes (Table 1).
25 Example 12: DTPA-arabinogalactan.
Purified arabinogalactan (20.0 g, 0.87 mmol) and the dianhydride of diethylenetriaminepentaacetic acid (DTPA) (2.15 g, 6.02 mmol~ were dissolved in dimethylsulfoxide (DMSO, 200 ml) at 60 C. After 0.5 hour, the clear solution 30 was added to HzO (ca. 500 ml) at 15 C. The solution was filtered on an Amicon YM3 and YMl ultrafiltration membranes t5,000 and 1,000 dalton cutoff, respectively) and washed with H2O (2 X 400 ml). The solution (70 ml) remaining on the membran~ was frozen and lyophilized~ Yield of white 35 powder was 18.8 g. The IR showed a band at 1734 cm~l (C=O).
Titration of an aqueous solution of the conjugate with 0.010 N NaOH indicated the presence of 0.117 milliequivalents DTPA
Wo 93/2~239 2 13 ~ ~ ~ r? PCT/US92/0~091 1 per gram DTPA-arabinogalactan.
Example 13: Gluta~yl-arabinogalactan.
Purified arabinogalactan (20.0 g, 0.87 mmol) and glutaric anhydride ~5.00 grams, 44 mm~l~ were dissolved in 5 DMSO (200 ml) at 60 C. The reaction mixture was cooled to ambient tempera~ure and allowed to react for 16 hours. The DMSO solution was added to H2O (200 ml), filtered on an Amicon YM3 ultrafiltration membrane and washed with H20 (2 times 300 ml). The solution remaining on the membrane was frozen and lyophilized. Yield of white powder: 18.5 g (lot number 2127-17~). IR (KBr): 1726 cm~~ (C=o).
- Example 14: S-2-t3 aminopropylamino) ethyl-thiopho~phate-dextran-arabinogalactan from thiopho~phorylated dextran.
PolythioPhos~horYlation of_dextran. Tén grams of :
15 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 20 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 25 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 30 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 35 mmole of thiophosphate is incorporated per gram of polysaccharide.
Svnthesis of 2-f3-aminoproPYlamino) ethvl bromide, W093~25239 PCT/US92/0509l 213~29~
dihydrobromide. Twenty three and six-tenths grams (200 mmole) of ice cold ~-(w-aminopropylamino) ethanol is added portionwise to 200 ml o~ ice cold 48-52% hydrobromic acid.
After stirring for 1 hour the reaction mixture is heated to 5 reflux for 1~-20 hours. The reaction mixture is vacuum dried to a reddish colored oil. The oil is titrated with 300 ml of acetone and left under refrigeration for 4 hours.
The mother liquor of acetone is decanted away from the gummy residue. The residue is dissolved with 75 ml of water and lO the resulting solution is added to 600 ml of cold acetone.
The crystalline precipitate is collected and then dissolved _ in boiling methanol. The resulting methanol solution is added to a ~0% mixture of ethyl ether and acetone (400 ml).
After cooling the mixture overnight the pure white crystals 15 are collected and vacuum dried. The melting point of the product is 205-206-C, as reported [Piper, J.R., et.al.
(1969), J. Med. Chem 12: 236-~43].
Reaction of polvthio~hosphorYlated dextran with 2-(w-aminopr~ylamino) ethyl bromide to form S-2-(3 20 aminopropylamino) ethyl-thiophosphate-dextran. Five mmoles of polythiophosphorylated dextran, sodium salt, is dissolved in lO ml of water. To the above solution is added 5.5 mmoles of 2-(3-aminopropylamino) ethyl bromide dihydrobromide dissolved in 10 mls of water. The clear 25 solution is stirred for four hours at room temperature. The resultinq turbid solution is added dropwise to rapidly stirred O-C ethanol. The resulting precipitate is collected by filtration. The product is washed with twice with 25 ml portions of warm (40-50-C) ethanol and vacuum dried.
The extent of thioalkylation is determined by a colorimetric analysis with ninhydrin.
Reaction of S-2-(3 aminopropYlamino) ethyl-thiophosphate-dextran with Arabinoqalactan-acylimidazole S-2-(3 aminopropylamino) ethyl-thiophosphate-dextran is 35 reacted with arabinogalactan-acylimidazole (Example 7) at 4-C for 16 hours. The product is isolated and purified by ultrafiltration using a YMlO filtration membrane.
-`~` W093/25239 2 1 3 ~ ~ 9 5 PCT/US92/05091 Example 15: Arabinogalactan-WR2721 from brominated nrabinogal~ctan.
Reduced arabinogalactan is bro~inated as described in Example 3. 2 grams of this brominated arabinogalactan is 5 added to 1 gram of WR2721 in 10 ml of 0.2M borate and the pH
adjusted to 8Ø 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 10 and dried. The final product is dissolved in 0.1 N HCl and titrated with 0.1 N NaOH. Using WR2721 as a reference for - the titration, 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 lS chromatography ~Amicon Cellufine GC200M) shows the major component has a molecul~r weight of about 25,000 daltons.
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 20 superparamagnetic iron-oxide colloid clearance, extending ~ the half-life of the colloid to 86 minutes (Table 1).
Ex~mple 16: Arabinogalactan-WR2721 from pho~phorylated arabinogalactan.
Arabinogalactan-phosphate (8 grams, example 9), 1.2 25 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 hour~. 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.
35 Characterization:
i. Molecular weight. Size exclusion chromatography (Amicon Cellufine GC200M) showed a single peak centered at W~93/25239 PCT/US92/05091 -~
213~2~5 22 minutes, similar to that observed for arabinogalactan-phosphate starting material. No evidence was seen of 1DW
molecular weight impurities.
ii. Analysis of sulfhydryl content. The product phosphate linked arabinogalactan-WR2721 is first hydrolyzed in 2M
trifluoroacetic acid for 1 hour at 120C. After neutralîzation, the sulfhydryl concPntration is measured by a colorimetric test using 5,5' bisdithio 2-nitrobenzoic acid. The amount of WR2721 on arabinogalactan was lO determined to be 0.063 milli-equivalents per gram of product.
- iii. Enzyme catalyzed hydrolysis. Both alkaline phosphatase (Biozyme Code ALPI-12G) at pH 8.0 and acid phosphatase (EC3.1.3.2, from potato) at pH 4.8 were found to 15 rapidly hydrolyze the phosphothioate ester and thus unblock the thiol. The rate of hydrolysis by the acid phosphatase was 0.1 micro-equi~alents phosphate/minute at 27-C, a rate which is close to that expected from the hydrolysis of p-nitrophenyl phosphate.
20 Example 17: Arabinogalactan-pep~tatin.
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 25 according to Example 2 (arabinogalactan-hydrazide) or example 5 or 7 (polylysine-arabinogalactan). Dissolve pepstatin A (250 mg) in 1 ml of dimethylformamide. Then add 50 mg 1-ethyl-3(3-dimethyl-aminopropyl)carbodiimide and 30 mg of N-hydroxy succimide. After the reaction has proceeded 30 at room temperature for 2 hours, add the mixture dropwise to 30 ml of 0.1 M sodium bicarbonate containing lOO mg of amino-arabinogalactan. Allow the resultant mixture is sit at room temperature for 2 h, then purify the product by ultrafiltration using a 10,000 dalton cutoff, and then by 35 cationic exchange chromatography.
-~ W093J2~239 ~1 3 5 2 ~ ~ PCT/US92/05091 Example 18: Carboxymethyl-arabinogalactan from reaction of bromo~cetic ~cid with arabinog~lactan Five grams of arabinogalactan is dissolved in 50 ml of 4N sodium hydroxide. To this is added lo grams of 5 bromoacetic acid, and the mixture heated at 80 D c for three hours. The reaction is terminated by cooling to room temperature then adjusting the pH to between 7.5 and 9 using concentrated hydrochloric acid. The product is isolated and purified by G-25 column chromatography and ultrafiltration 10 using an Amicon YM3 membrane. The extent of derivatization, ascertained by running the reaction with 14C labeled ^ bromoacetic acid and measuring the specific activity of the product by liquid scintillation counting, is about 5.2 milli-equivalents of carboxymethyl groups per gram of 15 product.
The activity of this arabinogalactan acetate was evaluated in the animal model as described above. A dose of lSO mg~kg was not an effective blocker of the superparamagnetic iron-oxide colloid, extending the half-20 life of the colloid only to 7.3 minutes, compared to 33.2minutes for underivatized arabinogalactan (Table 1).
Example 19: Carboxyethyl-arabinogalactan from reaction of 2-bromopropionic acid with arabinogalactan Fi~e grams of arabinogalactan is dissolved in 50 ml of 25 4N sodium NaOH. To this is added 11 grams of 2-bromopropionic acid, and the mixture heated at 80C for three hours. The reaction is terminated by cooling to room temperature, then adjusting the pH to between 7.5 and 9 using concentrated hydrochloric acid. The product is 30 isolated and purified by G-25 column chromatography and ;
ultrafiltration using an Amicon YM3 membrane. The extent of ~-derivatization, determined by acid/base titration, is about 1.3 milliequivalents propionate per gram of product. ;' The activity of arabinogalactan propionate was 35 evaluated in the animal model as described above. Use of 150 mg/kg of this substance showed it ineffective blocker in superparamagnetic iron-oxide colloid clearance assay, W093/25239 ~ PCT/US92~0~091 ~
~1352~S - ~6 -extending the half-life of the colloid to 40.8 minutes (Table 1).
Example 20: Arabinogalactan-W~2721 from thiopho~phorylated arnbinogal~ctan Thiophosphorylatlon of arabinoqalactan. 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 ~ath. To the cooled suspension is 10 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. After this time, the arabinogalactanyl thiophosphorodichloridate product is hydrolyzed by adding 50 15 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 g and 9.5 by the addition of 1 N sodium hydroxide.
20 The product is purified by ultra-filtration (50 ml to lO ml, ~- four cycles) using an Amicon YM3 (3000 dalton cutoff) ultrafiltration membrane. The final retentate is lyophilized to dryness.
Synthesis of 2-(3-aminopropylamino) eth~l bromide.
25 dihydrobromide. The synthesis of 2-(3-aminopropylamino) ethyl bromide, dihydrobromide is as described in Example 14~
Reaction of polythiophosPhorylated arabinoqalactan with 2-(w-aminopropylamino) ethvl bromide to form S-2-(3 aminopropylamino) ethvl-thio~hos~hate-arabinocalactan. Five 30 mmole of polythiophosphorylated arabinogalactan, sodium salt, is dissolved in 10 ml of water. To the above solution is added 5.5 mmole of 2-(3-aminopropylamino) ethyl bromide dihydrobromide dissolved in 10 ml of water. The clear solution is stirred for four hours at room temperature. The 35 resulting turbid solution is added dropwise to rapidly stirred O-C ethanol. The resulting precipitate is collected by filtration. The product is washed twice with 25 ml ,~ W093/25239 213 5 ~ 9 5 PCT/US92/05091 portions of warm (40-50 C) ethanol and vacuum dried.
Thioalkylation is confirmed by a colorimetric analysis with ninhydrin.
A second type of anti-viral agent that can be targeted with the teachings of the invention are antibodies. In this context antibodies may include polyclonal antibodies, monoclonal antibodies or antibody fragments. The natural 15 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 (l991), p. 28]. In particular hepatitis B virus replication 20 occurs within the hepatocytes of the liver and antibodies to viral antigens cannot directly bind the virus during this replication. If an antibody to a hepatitis B viral protein is conjugated to arabinogalactan, it will be targeted via the asialoglycoprotein receptor to the cytoplasm of 25 hepatocytes. Within the cytoplasm the antiviral antibody will bind replicating hepatitis B virus and become an effective therapeutic agent.
Some of the arabinogalactan derivatives described have ~, no known pharmacological activity, other than their ability 30 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 35 receptor, principally the hepatocytes of the liver. The carboxyl groups afforded by the succinyl-arabinogalactan, glutaryl-arabinogalactan and DTPA-arabinogalactan conjugates W093/2~239 213 S 2 n ~ PCT/US92/05091 (Examples 10-12) can be used to attach molecules through the use of carboniimides or other agents. The amino groups afforded by the arabinogalactan hydrazide (Example 3) or poly-L-lysine arabinogalactan (Examples 6, 8) can also be 5 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., Leonetti, J.P., Gagnor, C., Lemaitre, M., Lebleu, B., Nucleic Acids Res (1989) 17: 9341-15 50]. In addition to poly-L-Lysine, other polymeric molecules, such as dextrin, dextran, or albumin may be coupled to arabinogalactan.
In another embodiment, galactose oxidase treatment of arabinogalactan can be used to create aldehyde groups. The 20 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.
Similarly 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., He, S., Zhang, Z., 7th International Conference on Chemical 30 Modifiers of Cancer Treatment (1991), Clearwater, FA 338-340] or chemotherapy [Schein, P.S, International Conference on Chemical Modifiers of Cancer Treatment (1991), -Clearwater, FA 341-342]. The utility of WR2721 as a chemoprotectant has been objected to based on the lack of 35 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 W093/25239 PCTtUS92/05091 ~13 .~ 2 ~ ~
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 5 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, 10 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], 15 cationic aminothiols generally, glutathiols, and vitamin E
derivatives.
After synthesis, the interaction of the arabinogalactan derivative with the asialoglycoprotein receptor can be determined in vivo. The ability of a derivative to interact 20 with the asialoglycoprotein receptor is assessed by its ability to block the clearance of a substance recognized to ~ interact with the asiàloglycoprotein receptor based on --~ earlier work. An arabinogalactan coated superparamagnetic ~ iron oxide colloid interacts with this receptor and a ~ ~ .
25 quantitative assay for its clearance has been described below. In the absence of a blocking agent, 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 30 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.
To obtain the blood half-life a Sprague-Dawley rat (200-300 grams) is anesthetized (lO0 mg/kg of Inactin) and injected with a defined dose of a blocking agent, followed by an arabinogalactan coated superparamagnetic iron oxide at W093t25239 - 2 1 3 5 ~ 9 - PCT/USg2/05091 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/Tl the 5 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 10 a blocking agent (receptor binding activity). With antibodies and enzymes, covalent modification especially high levels of covalent modification, generally decreases or - destroys biological function. Thus it i~ surprising that arabinogalactan tolerates random modification with excellent 15 retention of its receptor-recognizing biological activity.
In fact 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 20 unknown. In contrast, 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 25 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.
If an arabinogalactan conjugate is inactive in the 30 blocking assay, i.e., does not prolong blood half-life, conditions used in conjugate synthesis can be adjusted to achieve a lower degree of modification. Alternatively, the $
modification strategy employed may be dropped altogether and a different procedure employed.
W093/25239 PC~/US92/05091 ~1352~
Interactions of arabinogalactan deri~atives with the asialoglycoprotei~ receptor Agent Dose Blocking Agent Half-life (mg/kg) (min) , _ _ None none 2.8 . _ _.
lactose 300 8.0 . ,. ~
arabinogalactan 150 33.2 phosphoryl- 150 51.0 arabinogalactan .
(Example 9) . .
succinyl 150 213 arabinogalactan (Example 11) _ arabinogalactan-AMP 15Q ~100 _ (Example 4) I
arabinogalactan- 150 86 WR2721 (Example 15) I _ arabinogalactan 150 7.3 acetate (Example 18) . .
arabinogalactan 150 40.8 propionate (Example 19) _ _ . ,.
The examples below demonstrate that arabinogalactan can be modified by the addition of phosphoryl, sulfhydryl, amino, carboxyl, halo, or acylimidazol groups, with receptor 30 binding activity being unaffected. The initial modification is performed on the hydroxyl groups on the arabinogalactan.
~- W093/25239 21~ S 2 ~J - PCT/US92/05091 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 5 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 10 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, l991).
Reagents used to couple proteins to solid phase amino or 15 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 àrabinogalactan to provide useful pharmaceutical agents are listed in Table 2.
_ r W093~2~239 ~ PCT/US92/05091 ,~
Therapeutic agents that have been or might be attached to ara~inogalactan or arabinogalactan derivatives , _ _ _ _ ~
Agent Class Reference I . . ,_ Ara-AMP antiviral activity Example 5 _ _ _ WR2721 chemo- and Example 14 radioprotective activity .
Pepstatin anti-inflammatory Example 17 activity : lO gene therapy Examples 6, 8 DNA .
,~ _ .
Antisense antiviral activity Examples 6, 8 nucleic acid : Antibody to antiviral activity : 15 hepatitis .~
. ~ Steriod ~: anti-inflammatory activity I _ _ _ _ Superoxide dismutase anti-inflammatory ¦ activity _ t Examples 2S '~
Ex~mple 1: Bromination of arabinogalactan.
The arabinogalactan (AG) used is from the Western Larch and chromatographs produre a single peak of about 20,000 daltons by size exclusion chromatography.
Ten grams of arabinogalactan are dissolved in 35 ml of - W093/25239 ~1 3 5 2 ~ ~ PCT/US92/0~091 a 7.1% (w/v) solution of Zn(BF4)2. Fifty ml of epibromohydrin is added and the solution stirred for so minutes at 100C. The bromo-arabinogalactan is precipitated in 150 ml cold (4-C) acetone, redissolved in water, and 5 precipitated in 150 ml cold ethanol. Analysis of the product for bromine showed 0.7 milliequivalents bromide per gram product.
Example 2: Treatment of arabinogala~tan with sodium borohydr~e.
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 NaBH4.
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 20 arabinogalactan produced no dye, indicating essentially complete reduction.
Example 3: ~ydrazino-arabinogalactan.
. .
Ten grams of reduced arabinogalactan (Example 2~ is dissolved in 35 ml of a 7.1% (w/v) aqueous solution of 25 Zn(BF~)2. Fifty ml of epibromohydrin is added and the solution stirred for 90 minutes at lOO 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 30 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. The hydrazido-arabinogalactan ^~
is precipitated in 150 ml cold (4-C) acetone, redissolved in water and precipitated in 150 ml cold ethanol. The product 35 hydrazide content is analyzed by acid-base titration and ~howed 0~25 milliequivalents hydrazide per gram of product.
W O 93/25239 PC~r/US92/05091 t -21352~5 Ex~mple 4: Arabino~alactan conjugated to adenosine 5~ monopho~phate ~AMP) One gram (2.9 mmoles) of adenosine 5'-monophosphate (AMP) is dissolved in 20 ml water with the addition of !-5 sodium bicarbonate powder. Arabinogalactan-hydrazide (0.6 g, example 2) is added and the pH adjusted to 7.5 with sodium hydroxide. One gram (5.2 mmoles) of 1-ethyl-3,4-- dimethylaminopropyl) carbodiimide is added and the reaction maintained at room temperature for 64 hours. The product is 10 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 15 buffer of 0.1 mM, pH 7.0 phosphate buffer at flow rate o.5 ml/min). A single broad pea~ 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 ~G-AMP product, based on the comparison of HPLC area under the 20 curve monitoring at 260 nm is 0.24, indicating approximately a 95% conversion of available hydrazide groups. The UV/VlS
, , 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 -~ 25 molecular weight approximately equivalent to underivatized arabinoga~lactan, about 20,000 daltons.
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 30 colloid, extending the half-life of the colloid to greater than 100 minutes (Table 1).
Ex~mple 5: Arabinogalactan conjugated to adenine 7 ar~binoside s~ monopho~phate (ARA-AMP).
One gram (2.9 mmoles) of adenine arabinoside 5'-35 monophosphate (ARA-AMP) is dissolved in 20 ml water with the addition of sodium bicarbonate powder. 0.6 grams of arabinogalactan-hydrazide (Example 2 above) is then added ' .
W093~25239 ~1 352~ - PCT/US92/0509l and the pH adjusted to 7.5 with the addition of sodium hydroxide. One gram (5.2 mg) of 1-ethyl-3,4-dimethylaminopropyl) carbodiimide is added and the reaction maintained at room temperature ~or 64 hours. The product is 5 purif~ed by ultrafiltration using an Amicon YM3 ultrafilter, and then further purified by precipitation fram ethanol and dried, yielding 280 mg of product. As with AG-AMP (Example 3), the strong cation exchange chromatography showed a single broad peak centered at 5.7 minutes and no measurable 10 residual unreacted ARA-AMP. The UV/VIS spectrum of AG-ARA-AMP was ~irtually identical to an ARA-AMP standard. Based _ on the area under the curve at an optical density of 260 nanometers and in comparison with an AMP standard, which is assumed to have the same extinction coefficient as ARA-AMP, 15 the product has 0.124 milli-equivalents of ARA-AMP per gram.
Ex~mple 6: Poly~L)lysyl-~rabinog~la~t~n ~prep~re~ by re~uctive ~mination).
Poly~L)lysine hydrochloride (1,000-4,000 daltons, 0.5 grams) is dissolved in 2 ml borate buffer (0.2M) and the pH
20 adjusted to 9.0 with sodium hydroxide. 100 mq arabinogalactan and 50 mg sodium cyanoborohydride is added.
~- and the reaction heated for 24 hours at 50-C. The product mixture is puriied using an Amicon YM3 ultrafilter. The retentate containing the polylysine-arabinogalactan 25 conjugate showed a positive ninhydrin test for amine and positive anthrone test for polysaccharide. The yield was 30 mg. Si~e exclusion high performance liquid chromatography (Amicon Cellufine GC200M) showed a product having a molecular weight approximately equal to the sum of molecular 30 weights of arabinogalactan and poly~L)lysine or about 25,000 daltons.
Ex~mple 7: Acylimidazole-arabinogalact~n.
Three grams of anhydrous arabinogalactan is suspended in 5 ml of anhydrous peroxide free dioxane. While stirring, 35 1.62 gm (lOmmole) of N,N'-carbonyl diimidazole, dissolved in 10 ml of dioxane, is added in a single portion. After stirring for 20 minutes the acylimidazol-arabinogalactan is W093/25~39 PCT/US92/05U91 ,~~
- ~8 -collected by fil~ration (medium frit). The praduct is washed with 25 ml of dioxane and refiltered. A second dioxane titration is performed. The product is next titrated with 25 ml of apf-diethyl ether and then vacuum 5 dried. Yield is 2.9 gm.
Ex~mple 8: Poly~L)ly~ine-arabi~ogalactan ~prepared rom acylimidazole-arabinogalactan).
One gram of acylimidazole-arabinogalactan (Example 7 and 0.2 grams poly~L)lysine (1,000-4,000 daltons) is 10 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 5C. The product is isolated first by precipitation in ethanol, and then purified using an Amicon YM10 ultrafilter. The retentate shows a positive 15 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.
The product poly(L)lysyl-arabinogalactan is analyzed by cation exchange chromatography HPLC (Rainin Synchropak 20 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.
The product, arabinogalactan-polylysine, elutes with a retention time of 3.9 minutes whereas unconjugated polylysine elutes with a retention time of 9.3 minutes.
25 Poly(L)lysine bound to arabinogalactan is verified by its UV
speatrum.
Example 9: Phosphoryl-arabinogalactan.
Two grams of arabinogalactan are dissolved in 20 ml formamide and 4 ml triethylamine. Ten grams polyphosphoric 30 acid are added and the reaction stirred for 1~ hours. The product is brought to pH 9 with 45~ NaOH and ultrafiltered in a SO ml stirred cell with a 3,000 molecular weight cutoff membrane ~Amicon), bringing the volume from 50 ml to 10 ml twice. The ultrafiltered product is precipitated into 500 35 ml cold acetone (4-C), redissolved, and precipitated in SOO
ml cold ethanol. The product showed 0.21 milli-equivalents of phosphate per gram of product both by acid base titration W093t25239 _ 2 1 3 5 2 ~ ;~ PCTiUS92/05091 -- lg --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).
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).
10 Example 10: Treatment of arabinogalactan with galactose oxida~e (GO).
- Ten grams of arabinogalactan is dissolved to a total volume of about 50 ml in O.1 M potassium phosphate buffer, pH = 6Ø To the resultant solution is added 225 units of 15 galactose oxidase dissolved in about 2 ml of the same buffer. The oxidation is allowed to proceed for 24 hours at room temperature. The H2O2 content is found to be about 3 mg/ml, as measured by peroxide test strips. A second addition of 22S units of GO is made to the reaction mixture.
20 After another 24 hour reaction period the peroxide content is found to be unchanged from the result of the first GO
treatment at about 3 mg/ml. Twenty milligrams of catalase ~--(dry solid) is added to decompose the peroxide. After standing at room temperature overnight the contents of the -- 25 flask are found to be free of peroxide. }
Product Purification. Ten grams of mixed bed resin, MB-l is added to the flask. After stirring for 30 minutes the solution is decanted into and passed through a short column containing an additional 5 grams of MB-l resin. The 30 pH neutral solution is found to be free of any protein amines by reaction with ninhydrin. The product is isolated by precipitation from 5-C cooled absolute ethanol. The precipitate is collected by filtration. The aldehyde content of this product is found to be between 3 and 5 times 35 greater than the aldehyde content of native arabinogalactan.
Yield is lO grams.
Determination of the Number of Aldehyde Groups: The 3---W093/25239 PCT/U~92/0509l ~
~13~
methyl-2-benzothiazolone hydrazone test for aldehyde was used to compare arabinogalactan starting material to poly-aldehydic arabinogalactan. Based on absorbance measured at 670 nm, this poly-aldehydic arabinogalactan has 0.34 milli-5 equivalents aldehyde per gram of ara~inogalactan.Ex~mple 11: ~uGcinyl-arabinogalactan.
Purified arabinogalactan (16.0 g, 0.70 mmol) and succinyl anhydride (10.0 g, 100 mmol) were dissolved in DMSO
(200 ml) at 60-C. After 1.0 hour, the clear, light yellow 10 solution was cooled to ambient temperature and allowed to stir for an additional 48 h. The DMSO solution was added to - H2O (200 ml), filtered on an Amicon YM3 ultrafiltration membrane and washed with H2O (3 times with 250 ml). The solution remaining on the membrane was frozen and 15 lyophilized. Yield of white powder: 20.~ g. IR (KBr):
1732 cm1 (C=O). Titration of an aqueous solution of the conjugate with G.01 N NaOH indicated the presence of 1.96 milli-equivalents succinate per gram of succinyl-arabinogalactan.
The activity of succinyl-arabinogalactan is 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 213 minutes (Table 1).
25 Example 12: DTPA-arabinogalactan.
Purified arabinogalactan (20.0 g, 0.87 mmol) and the dianhydride of diethylenetriaminepentaacetic acid (DTPA) (2.15 g, 6.02 mmol~ were dissolved in dimethylsulfoxide (DMSO, 200 ml) at 60 C. After 0.5 hour, the clear solution 30 was added to HzO (ca. 500 ml) at 15 C. The solution was filtered on an Amicon YM3 and YMl ultrafiltration membranes t5,000 and 1,000 dalton cutoff, respectively) and washed with H2O (2 X 400 ml). The solution (70 ml) remaining on the membran~ was frozen and lyophilized~ Yield of white 35 powder was 18.8 g. The IR showed a band at 1734 cm~l (C=O).
Titration of an aqueous solution of the conjugate with 0.010 N NaOH indicated the presence of 0.117 milliequivalents DTPA
Wo 93/2~239 2 13 ~ ~ ~ r? PCT/US92/0~091 1 per gram DTPA-arabinogalactan.
Example 13: Gluta~yl-arabinogalactan.
Purified arabinogalactan (20.0 g, 0.87 mmol) and glutaric anhydride ~5.00 grams, 44 mm~l~ were dissolved in 5 DMSO (200 ml) at 60 C. The reaction mixture was cooled to ambient tempera~ure and allowed to react for 16 hours. The DMSO solution was added to H2O (200 ml), filtered on an Amicon YM3 ultrafiltration membrane and washed with H20 (2 times 300 ml). The solution remaining on the membrane was frozen and lyophilized. Yield of white powder: 18.5 g (lot number 2127-17~). IR (KBr): 1726 cm~~ (C=o).
- Example 14: S-2-t3 aminopropylamino) ethyl-thiopho~phate-dextran-arabinogalactan from thiopho~phorylated dextran.
PolythioPhos~horYlation of_dextran. Tén grams of :
15 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 20 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 25 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 30 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 35 mmole of thiophosphate is incorporated per gram of polysaccharide.
Svnthesis of 2-f3-aminoproPYlamino) ethvl bromide, W093~25239 PCT/US92/0509l 213~29~
dihydrobromide. Twenty three and six-tenths grams (200 mmole) of ice cold ~-(w-aminopropylamino) ethanol is added portionwise to 200 ml o~ ice cold 48-52% hydrobromic acid.
After stirring for 1 hour the reaction mixture is heated to 5 reflux for 1~-20 hours. The reaction mixture is vacuum dried to a reddish colored oil. The oil is titrated with 300 ml of acetone and left under refrigeration for 4 hours.
The mother liquor of acetone is decanted away from the gummy residue. The residue is dissolved with 75 ml of water and lO the resulting solution is added to 600 ml of cold acetone.
The crystalline precipitate is collected and then dissolved _ in boiling methanol. The resulting methanol solution is added to a ~0% mixture of ethyl ether and acetone (400 ml).
After cooling the mixture overnight the pure white crystals 15 are collected and vacuum dried. The melting point of the product is 205-206-C, as reported [Piper, J.R., et.al.
(1969), J. Med. Chem 12: 236-~43].
Reaction of polvthio~hosphorYlated dextran with 2-(w-aminopr~ylamino) ethyl bromide to form S-2-(3 20 aminopropylamino) ethyl-thiophosphate-dextran. Five mmoles of polythiophosphorylated dextran, sodium salt, is dissolved in lO ml of water. To the above solution is added 5.5 mmoles of 2-(3-aminopropylamino) ethyl bromide dihydrobromide dissolved in 10 mls of water. The clear 25 solution is stirred for four hours at room temperature. The resultinq turbid solution is added dropwise to rapidly stirred O-C ethanol. The resulting precipitate is collected by filtration. The product is washed with twice with 25 ml portions of warm (40-50-C) ethanol and vacuum dried.
The extent of thioalkylation is determined by a colorimetric analysis with ninhydrin.
Reaction of S-2-(3 aminopropYlamino) ethyl-thiophosphate-dextran with Arabinoqalactan-acylimidazole S-2-(3 aminopropylamino) ethyl-thiophosphate-dextran is 35 reacted with arabinogalactan-acylimidazole (Example 7) at 4-C for 16 hours. The product is isolated and purified by ultrafiltration using a YMlO filtration membrane.
-`~` W093/25239 2 1 3 ~ ~ 9 5 PCT/US92/05091 Example 15: Arabinogalactan-WR2721 from brominated nrabinogal~ctan.
Reduced arabinogalactan is bro~inated as described in Example 3. 2 grams of this brominated arabinogalactan is 5 added to 1 gram of WR2721 in 10 ml of 0.2M borate and the pH
adjusted to 8Ø 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 10 and dried. The final product is dissolved in 0.1 N HCl and titrated with 0.1 N NaOH. Using WR2721 as a reference for - the titration, 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 lS chromatography ~Amicon Cellufine GC200M) shows the major component has a molecul~r weight of about 25,000 daltons.
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 20 superparamagnetic iron-oxide colloid clearance, extending ~ the half-life of the colloid to 86 minutes (Table 1).
Ex~mple 16: Arabinogalactan-WR2721 from pho~phorylated arabinogalactan.
Arabinogalactan-phosphate (8 grams, example 9), 1.2 25 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 hour~. 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.
35 Characterization:
i. Molecular weight. Size exclusion chromatography (Amicon Cellufine GC200M) showed a single peak centered at W~93/25239 PCT/US92/05091 -~
213~2~5 22 minutes, similar to that observed for arabinogalactan-phosphate starting material. No evidence was seen of 1DW
molecular weight impurities.
ii. Analysis of sulfhydryl content. The product phosphate linked arabinogalactan-WR2721 is first hydrolyzed in 2M
trifluoroacetic acid for 1 hour at 120C. After neutralîzation, the sulfhydryl concPntration is measured by a colorimetric test using 5,5' bisdithio 2-nitrobenzoic acid. The amount of WR2721 on arabinogalactan was lO determined to be 0.063 milli-equivalents per gram of product.
- iii. Enzyme catalyzed hydrolysis. Both alkaline phosphatase (Biozyme Code ALPI-12G) at pH 8.0 and acid phosphatase (EC3.1.3.2, from potato) at pH 4.8 were found to 15 rapidly hydrolyze the phosphothioate ester and thus unblock the thiol. The rate of hydrolysis by the acid phosphatase was 0.1 micro-equi~alents phosphate/minute at 27-C, a rate which is close to that expected from the hydrolysis of p-nitrophenyl phosphate.
20 Example 17: Arabinogalactan-pep~tatin.
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 25 according to Example 2 (arabinogalactan-hydrazide) or example 5 or 7 (polylysine-arabinogalactan). Dissolve pepstatin A (250 mg) in 1 ml of dimethylformamide. Then add 50 mg 1-ethyl-3(3-dimethyl-aminopropyl)carbodiimide and 30 mg of N-hydroxy succimide. After the reaction has proceeded 30 at room temperature for 2 hours, add the mixture dropwise to 30 ml of 0.1 M sodium bicarbonate containing lOO mg of amino-arabinogalactan. Allow the resultant mixture is sit at room temperature for 2 h, then purify the product by ultrafiltration using a 10,000 dalton cutoff, and then by 35 cationic exchange chromatography.
-~ W093J2~239 ~1 3 5 2 ~ ~ PCT/US92/05091 Example 18: Carboxymethyl-arabinogalactan from reaction of bromo~cetic ~cid with arabinog~lactan Five grams of arabinogalactan is dissolved in 50 ml of 4N sodium hydroxide. To this is added lo grams of 5 bromoacetic acid, and the mixture heated at 80 D c for three hours. The reaction is terminated by cooling to room temperature then adjusting the pH to between 7.5 and 9 using concentrated hydrochloric acid. The product is isolated and purified by G-25 column chromatography and ultrafiltration 10 using an Amicon YM3 membrane. The extent of derivatization, ascertained by running the reaction with 14C labeled ^ bromoacetic acid and measuring the specific activity of the product by liquid scintillation counting, is about 5.2 milli-equivalents of carboxymethyl groups per gram of 15 product.
The activity of this arabinogalactan acetate was evaluated in the animal model as described above. A dose of lSO mg~kg was not an effective blocker of the superparamagnetic iron-oxide colloid, extending the half-20 life of the colloid only to 7.3 minutes, compared to 33.2minutes for underivatized arabinogalactan (Table 1).
Example 19: Carboxyethyl-arabinogalactan from reaction of 2-bromopropionic acid with arabinogalactan Fi~e grams of arabinogalactan is dissolved in 50 ml of 25 4N sodium NaOH. To this is added 11 grams of 2-bromopropionic acid, and the mixture heated at 80C for three hours. The reaction is terminated by cooling to room temperature, then adjusting the pH to between 7.5 and 9 using concentrated hydrochloric acid. The product is 30 isolated and purified by G-25 column chromatography and ;
ultrafiltration using an Amicon YM3 membrane. The extent of ~-derivatization, determined by acid/base titration, is about 1.3 milliequivalents propionate per gram of product. ;' The activity of arabinogalactan propionate was 35 evaluated in the animal model as described above. Use of 150 mg/kg of this substance showed it ineffective blocker in superparamagnetic iron-oxide colloid clearance assay, W093/25239 ~ PCT/US92~0~091 ~
~1352~S - ~6 -extending the half-life of the colloid to 40.8 minutes (Table 1).
Example 20: Arabinogalactan-W~2721 from thiopho~phorylated arnbinogal~ctan Thiophosphorylatlon of arabinoqalactan. 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 ~ath. To the cooled suspension is 10 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. After this time, the arabinogalactanyl thiophosphorodichloridate product is hydrolyzed by adding 50 15 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 g and 9.5 by the addition of 1 N sodium hydroxide.
20 The product is purified by ultra-filtration (50 ml to lO ml, ~- four cycles) using an Amicon YM3 (3000 dalton cutoff) ultrafiltration membrane. The final retentate is lyophilized to dryness.
Synthesis of 2-(3-aminopropylamino) eth~l bromide.
25 dihydrobromide. The synthesis of 2-(3-aminopropylamino) ethyl bromide, dihydrobromide is as described in Example 14~
Reaction of polythiophosPhorylated arabinoqalactan with 2-(w-aminopropylamino) ethvl bromide to form S-2-(3 aminopropylamino) ethvl-thio~hos~hate-arabinocalactan. Five 30 mmole of polythiophosphorylated arabinogalactan, sodium salt, is dissolved in 10 ml of water. To the above solution is added 5.5 mmole of 2-(3-aminopropylamino) ethyl bromide dihydrobromide dissolved in 10 ml of water. The clear solution is stirred for four hours at room temperature. The 35 resulting turbid solution is added dropwise to rapidly stirred O-C ethanol. The resulting precipitate is collected by filtration. The product is washed twice with 25 ml ,~ W093/25239 213 5 ~ 9 5 PCT/US92/05091 portions of warm (40-50 C) ethanol and vacuum dried.
Thioalkylation is confirmed by a colorimetric analysis with ninhydrin.
Claims (69)
1. A carrier for forming a complex with a therapeutic agent for delivery thereof to a cell receptor located on the surface of a target tissue comprising:
arabinogalactan modified at a site by a functional residue to produce a derivative in a manner that preserves the useful affinity of the derivative for the cell receptor.
arabinogalactan modified at a site by a functional residue to produce a derivative in a manner that preserves the useful affinity of the derivative for the cell receptor.
2. A carrier according to claim 1, wherein the site of modification is a hydroxyl group on the arabinogalactan.
3. A carrier according to claim 1, wherein the arabinogalactan is modified at a plurality of sites.
4. A carrier according to claim 2, wherein the arabinogalactan is modified at a plurality of sites and the number of functional residues is no less than one equivalent per mole of arabinogalactan and no more than the number of hydroxyl groups on arabinogalactan per mole of arabinogalactan.
5. A carrier according to claim 1, wherein the functional residue is selected from the group consisting of phosphoryl, sulfhydryl, amino, halo, acylimidizole and carboxyl groups.
6. A carrier according to claim 2, wherein the functional residue is selected from the group consisting of phosphoryl, sulfhydryl, amino, halo, acylimidazol and carboxyl groups.
7. A carrier according to claim 1, wherein the functional residue is a polymeric molecule.
8. A carrier according to claim 2, wherein the functional residue is a polymeric molecule.
9. A carrier according to claim 7, wherein the functional residue is a polymeric molecule selected from the group consisting of dextran, dextrin, albumin and poly-L-lysine.
10. A carrier according to claim 8, wherein the functional residue is a polymeric molecule selected from the group consisting of dextran, dextrin, albumin and poly-L-lysine.
11. A complex for delivery of a therapeutic agent to a cell receptor located on the surface of a target tissue comprising:
arabinogalactan modified by a functional residue to produce a derivative in a manner that preserves the useful affinity of the derivative for the cell receptor; and a therapeutic agent associated with the derivative so as to form a complex.
arabinogalactan modified by a functional residue to produce a derivative in a manner that preserves the useful affinity of the derivative for the cell receptor; and a therapeutic agent associated with the derivative so as to form a complex.
12. A complex according to claim 11, wherein the therapeutic agent is an antiviral agent.
13. A complex according to claim 12, wherein the antiviral agent is selected from the group consisting of acyclovir, ARA-AMP and ARA-A.
14. A complex according to claim 11, wherein the therapeutic agent is a radioprotective agent.
15. A complex according to claim 14, wherein the radioprotective agent is S-2-(3 aminopropylamino)ethyl phosphoric acid.
16. A complex according to claim 11, wherein the therapeutic agent is a chemoprotective agent.
17. A complex according to claim 16, wherein the chemoprotective agent is S-2-(3 aminopropylamino) ethyl phosphoric acid.
18. A complex according to claim 16, wherein the therapeutic agent is a free radical scavenger.
19. A complex according to claim 18, wherein the free radical scavenger is selected the group consisting of a melanin, Trolox, cysteamine derivatives, cationic aminothiols and vitamin E derivatives.
20. A complex according to claim 11, wherein the therapeutic agent is a polypeptide.
21. A complex according to claim 20, wherein the polypeptide is pepstatin.
22. A complex according to claim 20, wherein the polypeptide is an enzyme.
23. A complex according to claim 22, wherein the enzyme is superoxide dismutase.
24. A complex according to claim 11, wherein the therapeutic agent is selected from the group consisting of a polyclonal antibody, a monoclonal antibody and an antibody fragment.
25. A complex according to claim 11, wherein the therapeutic agent is a DNA molecule.
26. A complex according to claim 25, wherein the DNA
molecule is an antisense molecule.
molecule is an antisense molecule.
27. A complex according to claim 11, wherein the therapeutic agent is a steroid.
28. A method of delivering a therapeutic agent to a cell receptor comprising:
modifying arabinogalactan at a site by a functional residue to produce a derivative in a manner that preserves the useful affinity of the derivative for the cell receptor;
and associating a therapeutic agent with the derivative so as to form a complex.
modifying arabinogalactan at a site by a functional residue to produce a derivative in a manner that preserves the useful affinity of the derivative for the cell receptor;
and associating a therapeutic agent with the derivative so as to form a complex.
29. A method of according to claim 8, wherein the site is a hydroxyl group on the arabinogalactan.
30. A method of according to claim 25, wherein the arabinogalactan includes modifying arabinogalactan at a plurality of sites.
31. A method according to claim 28, wherein the step of modifying arabinogalactan includes modifying arabinogalactan at a plurality of sites wherein the number of functional residues is no less than one equivalent per mole of arabinogalactan and no more than the number of hydroxyl groups on arabinogalactan per mole of arabinogalactan.
32. A method according to claim 28, wherein the step of modifying the functional residue includes selecting the functional residue from the group consisting of phosphoryl, sulfhydryl, amino, halo! acylimidazol and carboxyl groups.
33. A method according to claim 29, wherein the step of modifying the functional residue includes selecting the functional residue from the group consisting of phosphoryl, sulfhydryl, amino, halo, acylimidazol and carboxyl groups.
34. A method according to claim 28, wherein the functional residue is a polymeric molecule.
35. A method according to claim 28, wherein the therapeutic agent is a polypeptide.
36. A method according to claim 35, wherein the polypeptide is an enzyme.
37. A method according to claim 35, wherein the polypeptide is superoxide dismutase.
38. A method according to claim 35, wherein the polypeptide is pepstatin.
39. A method according to claim 35, wherein the polypeptide is selected from the group consisting of a monoclonal antibody, a polyclonal antibody and an antibody fragment.
40. A method according to claim 28, wherein the therapeutic agent is a DNA molecule.
41. A method according to claim 40, wherein the DNA
molecule is an antisense molecule.
molecule is an antisense molecule.
42. A method according to claim 28, wherein the therapeutic agent is a steroid.
43. A method according to claim 28, wherein the therapeutic agent is selected from the group consisting of an antiviral agent, a radioprotective agent and a chemoprotective agent.
44. A method according to claim 43, wherein the radioprotective agent is S-2-(3 aminopropylamino) ethyl-thiophophoric acid.
45. A method according to claim 43, wherein the chemoprotective agent is S-2-(3 aminopropylamino) ethyl-thiophophoric acid.
46. A method according to claim 43, wherein the chemoprotective agent is a free radical scavenger.
47. A method according to claim 46, wherein the free radical scavenger is selected from a group consisting of a melanin, Trolox, cysteamine derivatives, cationic aminothiols and vitamin E derivatives.
48. A method according to claim 43, wherein the antiviral agent is selected from the group consisting of acyclovir, ARA-AMP and ARA-A.
49. A method according to claim 28, further comprising:
administering an effective amount of the complex to an organism having the cell receptor.
administering an effective amount of the complex to an organism having the cell receptor.
50. A method according to claim 29, further comprising:
administering an effective amount of the complex to an organism having the cell receptor.
administering an effective amount of the complex to an organism having the cell receptor.
51. A method according to claim 30, further comprising:
administering an effective amount of the complex to an organism having the cell receptor.
administering an effective amount of the complex to an organism having the cell receptor.
52. A method according to claim 31, further comprising:
administering an effective amount of the complex to an organism having the cell receptor.
administering an effective amount of the complex to an organism having the cell receptor.
53. A method according to claim 32, further comprising:
administering an effective amount of the complex to an organism having the cell receptor.
administering an effective amount of the complex to an organism having the cell receptor.
54. A method according to claim 34, further comprising:
administering an effective amount of the complex to an organism having the cell receptor.
administering an effective amount of the complex to an organism having the cell receptor.
55. A method according to claim 35, further comprising:
administering an effective amount of the complex to an organism having the cell receptor.
administering an effective amount of the complex to an organism having the cell receptor.
56. A method according to claim 40, further comprising:
administering an effective amount of the complex to an organism having the cell receptor.
administering an effective amount of the complex to an organism having the cell receptor.
57. A method according to claim 42, further comprising:
administering an effective amount of the complex to an organism having the cell receptor.
administering an effective amount of the complex to an organism having the cell receptor.
58. A method according to claim 43, further comprising:
administering an effective amount of the complex to an organism having the cell receptor.
administering an effective amount of the complex to an organism having the cell receptor.
59. A method according to claim 46, further comprising:
administering an effective amount of the complex to an organism having the cell receptor.
administering an effective amount of the complex to an organism having the cell receptor.
60. A process for making a carrier that is suitable for forming a complex with a therapeutic agent for delivery thereof to a cell receptor, comprising:
providing arabinogalactan;
treating the arabinogalactan with an agent suitable for modifying the aldehyde content thereof to produce a derivative that preserves the useful affinity of the derivative with the cell receptor.
providing arabinogalactan;
treating the arabinogalactan with an agent suitable for modifying the aldehyde content thereof to produce a derivative that preserves the useful affinity of the derivative with the cell receptor.
61. A process according to claim 60, wherein the agent is suitable for increasing the aldehyde content of arabinogalactan.
62. A process according to claim 61, wherein the agent is galactose oxidase.
63. A process according to claim 62, wherein the step of treating arabinogalactan includes the step of oxidizing arabinogalactan by galactose oxidase so as to contain at least one additional aldehyde and less than about 1.2 milli-equivalents of aldehydes per gram of polysaccharide.
64. A process according to claim 63, wherein the agent is suitable for decreasing the aldehyde content of arabinogalactan.
65. A process according to claim 60, wherein the agent is a reducing agent containing borohydride.
66. A process according to claim 65, wherein the borohydride is selected from the group consisting of sodium borohydride and sodium cyanoborohydride.
67. A process according to claim 60, wherein the step of treating arabinogalactan includes hydrolyzing arabinogalactan by an endo-beta galactosidase enzyme.
68. A process according to claim 60, wherein the step of treating arabinogalactan includes reacting the arabinogalactan with anhydrides selected from the group consisting of succinate, glutarate and diethylenepentaacetic acid.
69. A process according to claim 60, wherein the step of treating arabinogalactan includes the step of reacting the arabinogalactan with a primary amine to produce a Schiff's base and treating the product with a reducing agent.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CA002135295A CA2135295A1 (en) | 1992-06-17 | 1992-06-17 | Arabinogalactan derivatives and uses thereof |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
PCT/US1992/005091 WO1993025239A1 (en) | 1992-06-17 | 1992-06-17 | Arabinogalactan derivatives and uses thereof |
CA002135295A CA2135295A1 (en) | 1992-06-17 | 1992-06-17 | Arabinogalactan derivatives and uses thereof |
Publications (1)
Publication Number | Publication Date |
---|---|
CA2135295A1 true CA2135295A1 (en) | 1993-12-23 |
Family
ID=4154623
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CA002135295A Abandoned CA2135295A1 (en) | 1992-06-17 | 1992-06-17 | Arabinogalactan derivatives and uses thereof |
Country Status (5)
Country | Link |
---|---|
EP (1) | EP0646018A1 (en) |
JP (1) | JPH07507794A (en) |
CA (1) | CA2135295A1 (en) |
NO (1) | NO944838L (en) |
WO (1) | WO1993025239A1 (en) |
Families Citing this family (21)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5554386A (en) * | 1986-07-03 | 1996-09-10 | Advanced Magnetics, Inc. | Delivery of therapeutic agents to receptors using polysaccharides |
US5490991A (en) * | 1986-07-03 | 1996-02-13 | Advanced Magnetics, Inc. | Directed delivery of radioprotectants using a receptor specific carrier |
US5728518A (en) * | 1994-01-12 | 1998-03-17 | The Immune Response Corporation | Antiviral poly-and oligonucleotides |
FR2716625B1 (en) * | 1994-02-25 | 1996-04-26 | Gouchet Franck Arno | Preparation and use of pharmaceutical and cosmetic forms containing inclusion compounds with cysteamine cyclodextrins, cysteamine salts, or cysteamine derivatives, in salt form or not. |
US5567685A (en) * | 1994-08-16 | 1996-10-22 | Yissum Research Development Company Of The Hebrew University Of Jerusalem | Water-Soluble polyene conjugate |
US5897987A (en) * | 1996-03-25 | 1999-04-27 | Advanced Reproduction Technologies, Inc. | Use of arabinogalactan in cell cryopreservation media |
US6303584B1 (en) * | 1996-11-20 | 2001-10-16 | The University Of Montana | Water soluble lipidated arabinogalactan |
US6011008A (en) * | 1997-01-08 | 2000-01-04 | Yissum Research Developement Company Of The Hebrew University Of Jerusalem | Conjugates of biologically active substances |
ATE466948T1 (en) | 1997-03-11 | 2010-05-15 | Univ Minnesota | DNA-BASED TRANSPOSON SYSTEM FOR INTRODUCING NUCLIC ACID INTO A CELL'S DNA |
US6929936B1 (en) * | 1997-07-18 | 2005-08-16 | Danisco A/S | Composition comprising an enzyme having galactose oxidase activity and use thereof |
CA2330318A1 (en) * | 1998-04-27 | 1999-11-04 | Larex, Inc. | Derivatives of arabinogalactan and compositions including the same |
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 |
IL131074A0 (en) * | 1999-07-23 | 2001-03-19 | Polygene Ltd | A biodegradable polycation composition for delivery of an anionic macromolecule |
WO2001089579A2 (en) * | 2000-05-19 | 2001-11-29 | Regents Of The University Of Minnesota | Composition for delivery of compounds to cells |
WO2003089618A2 (en) | 2002-04-22 | 2003-10-30 | Regents Of The University Of Minnesota | Transposon system and methods of use |
ITMI20040928A1 (en) | 2004-05-07 | 2004-08-07 | Uni Di Bologna Dipartiment O D | PROCEDURE FOR THE PREPARATION OF DOXORUBICIN CONJUGATES WITH LACTOSAMINATED HUMAN ALBUMIN |
ITMI20051743A1 (en) * | 2005-09-20 | 2007-03-21 | Uni Di Bologna Dipartimento Di Patologia Spa | USE OF DOXORUBICINE CONJUGIES WITH LATTOSAMINATED ALBUMIN |
RU2359975C2 (en) * | 2007-04-04 | 2009-06-27 | Институт нефтехимии и катализа РАН | Method for production of modified arabinogalactan |
CN102399238B (en) * | 2011-12-21 | 2013-06-12 | 开封明仁药业有限公司 | Preparation method for amifostine |
RU2521649C1 (en) * | 2013-03-04 | 2014-07-10 | Федеральное Государственное Бюджетное Учреждение Науки Институт Химии И Химической Технологии Сибирского Отделения Российской Академии Наук (Иххт Со Ран) | Method of producing sulphated arabinogalactan derivatives |
US10994013B2 (en) | 2013-04-24 | 2021-05-04 | Temple University—Of the Commonwealth System of Higher Education | Solid dosage form containing arabinogalactan |
Family Cites Families (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CA1184492A (en) * | 1981-01-14 | 1985-03-26 | Masataka Morishita | Preparation having excellent absorption property |
JPH0816118B2 (en) * | 1988-02-03 | 1996-02-21 | 呉羽化学工業株式会社 | Antiviral agent |
EP0381742B1 (en) * | 1988-08-04 | 1996-06-19 | Advanced Magnetics Incorporated | Receptor mediated endocytosis type mri contrast agents |
-
1992
- 1992-06-17 WO PCT/US1992/005091 patent/WO1993025239A1/en not_active Application Discontinuation
- 1992-06-17 CA CA002135295A patent/CA2135295A1/en not_active Abandoned
- 1992-06-17 JP JP6501422A patent/JPH07507794A/en active Pending
- 1992-06-17 EP EP92914217A patent/EP0646018A1/en not_active Withdrawn
-
1994
- 1994-12-14 NO NO944838A patent/NO944838L/en unknown
Also Published As
Publication number | Publication date |
---|---|
NO944838D0 (en) | 1994-12-14 |
EP0646018A1 (en) | 1995-04-05 |
JPH07507794A (en) | 1995-08-31 |
NO944838L (en) | 1995-02-17 |
WO1993025239A1 (en) | 1993-12-23 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US5478576A (en) | Arabinogalactan derivatives and uses thereof | |
CA2135295A1 (en) | Arabinogalactan derivatives and uses thereof | |
US5554386A (en) | Delivery of therapeutic agents to receptors using polysaccharides | |
Groman et al. | Arabinogalactan for hepatic drug delivery | |
US5433955A (en) | Site specific in vivo activation of therapeutic drugs | |
Takagishi et al. | Macromolecule‐small molecule interactions; introduction of additional binding sites in polyethyleneimine by disulfide cross–linkages | |
Aplin et al. | Preparation, Properties, and Applications of Carbohydrate Conjugates of Proteins and Lipid | |
EP2173383B1 (en) | Hydrolysable polymeric fmoc-linker | |
CA2506842A1 (en) | Amplification of biotin-mediated targeting | |
KR20060085329A (en) | Polysialic acid derivatives | |
EP0377613B1 (en) | Conjugates of superoxide dismutase | |
Tsukada et al. | An anti-α-fetoprotein antibody-daunorubicin conjugate with a novel poly-L-glutamic acid derivative as intermediate drug carrier | |
Molteni | [22] Dextran and inulin conjugates as drug carriers | |
FI97692B (en) | Process for preparing a therapeutically useful conjugate of an activator and a targeting moiety | |
Enriquez et al. | Conjugation of adenine arabinoside 5'-monophosphate to arabinogalactan: synthesis, characterization, and antiviral activity | |
WO1998056424A1 (en) | Biologically active materials | |
Orlando | Modification of proteins and low molecular weight substances with hydroxyethyl starch (HES) | |
CA2023339A1 (en) | Method for producing synthetic n-linked glycoconjugates | |
KR950007216B1 (en) | Preparation method of ribosome-inactivating glycoproteins, modified by oxidation of their osidic units and reduction | |
SK9785A3 (en) | Polymeric remedy and preparation method thereof | |
EP0628315A1 (en) | Lectin complex and probe and method for making same | |
WO1997022879A1 (en) | Modified avidin-type molecules as targeting agents for the liver and cells of the reticuloendothelial system | |
Orlando | Modification of proteins and low molecular weight substances with hydroxyethyl starch (HES): HESylation-a new technology for polymer conjugation to biologically active molecules | |
Diedrich et al. | Phloretin and Phlorizin Derivative Containing Compounds |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
EEER | Examination request | ||
FZDE | Dead |