EP0722326A1 - Maltooligosaccharides fortement sulfatees presentant des proprietes semblables a l'heparine - Google Patents

Maltooligosaccharides fortement sulfatees presentant des proprietes semblables a l'heparine

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Publication number
EP0722326A1
EP0722326A1 EP94931314A EP94931314A EP0722326A1 EP 0722326 A1 EP0722326 A1 EP 0722326A1 EP 94931314 A EP94931314 A EP 94931314A EP 94931314 A EP94931314 A EP 94931314A EP 0722326 A1 EP0722326 A1 EP 0722326A1
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EP
European Patent Office
Prior art keywords
highly sulfated
sulfated
composition
heparin
compositions
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.)
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Application number
EP94931314A
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German (de)
English (en)
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EP0722326A4 (fr
Inventor
Peter Fugedi
David John Tyrrell
Robert James Tressler
Robert Joseph Stack
Masayuki Ishihara
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Glycomed Inc
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Glycomed Inc
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Publication of EP0722326A1 publication Critical patent/EP0722326A1/fr
Publication of EP0722326A4 publication Critical patent/EP0722326A4/fr
Withdrawn legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/737—Sulfated polysaccharides, e.g. chondroitin sulfate, dermatan sulfate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H11/00—Compounds containing saccharide radicals esterified by inorganic acids; Metal salts thereof

Definitions

  • the invention relates to a novel class of highly sulfated maltooligosaccharides having heparin-like activity, and to methods for using these oligosaccharides, alone or in combination with a chemotherapeutic drug, to treat certain diseases.
  • Heparin and other naturally occurring glycosaminoglycans possess medically useful properties.
  • Recently, several low molecular weight heparins from various chemical or enzymatic depolymerization processes have been developed for clinical use. Thomas et al., Thrombos. Res. (1982) 28:343-350; Walenga et al., Thrombos. Res. (1986) 43:243-248; Koller et al., Thrombos Haemostas. (1986) 56:243-246.
  • the involvement of heparin or heparan sulfate or degradation products thereof in smooth muscle proliferation has been recognized for some time.
  • Heparin and heparan sulfate can slow or arrest the vascular proliferation associated with injury described hereinabove (Clowes, A.W., et al., Nature (1977) 265:625-626).
  • the effect of heparan sulfate and heparin on smooth muscle proliferation is also described by Marcum, J.A., et al. in Biology of Proteoqlvcan, Academic Press (1987) pp. 301-343.
  • heparin The inhibition of vascular smooth muscle cell growth by heparin was further described by Castellot, J.J., Jr., et al., J Biol Chem (1982) 257:11256-11260, and the effect of heparin on vascular smooth muscle cell growth in fetal tissue was described by Benitz, W.E., et al., J Cell Phvsiol (1986) 127:1-7.
  • the effect of heparin as an inhibitor of both pericyte and smooth muscle cell proliferation was shown by Orlidge, A., et al., Microvascular Research (1986) 31 :41-53, and these authors further showed that chondroitin sulfate, and dermatan sulfate do not have this effect.
  • Heparins and certain low molecular weight heparins have certain disadvantages for medical applications. Generally, both compositions exhibit anticoagulant activity, and thus must be administered with considerable medical supervision. Recently, however, non-anticoagulant heparins have been developed to circumvent this problem and are now undergoing clinical trials. Secondly, heparins are of natural origin, generally being purified from animals. As such, small amounts of the glycosaminoglycans may cause anaphylactic reactions, a decrease in the number of thrombocytes, thrombosis and embolism. Therefore, the preparation or identification of synthetic agents possessing properties comparable to heparin is desired.
  • U.S. Patent No. 4,066,829 discloses complement system-modulating activity for sulfated maltodextrin polymers of undisclosed molecular weight. Similar complement system-modulating activity for 4-O-polyhexose-thio-arylene sulfate derivatives is disclosed in U.S. Patent No. 4,470,976 and U.S. Patent No. 4,435,387.
  • the hexose polymers described are substituted disaccharides.
  • EPA 0338092 describes alkali metal or alkaline earth metal salts of a sulfated linear polymer of 4 to 10 D-glucose units linked ⁇ -1 ,4. These compositions are claimed to have anti-HIV activity.
  • the instant invention provides highly sulfated maltooligosaccharide compositions having therapeutic and/or prophylactic properties alone or in combination with a chemotherapeutic drug.
  • the compositions have properties similar to heparin.
  • the invention provides highly sulfated maltooligosaccharide compositions that are effective inhibitors of smooth muscle cell proliferation.
  • a third aspect of the invention is the description of highly sulfated maltooligosaccharide compositions that have the following structural formula:
  • each R 1 independently represents an alkyl, aryl, or aralkyl group, a reduced or oxidized glucose unit, S0 3 M, or H;
  • R 2 represents a S0 3 M group or H;
  • M represents a biologically acceptable cation
  • n represents an integer from 1 to 9; with the proviso that at least 50% of R 2 groups are S0 3 M.
  • a fourth aspect of the invention is the description of preferred highly sulfated maltooligosaccharide compositions including sulfated maltotetraose, sulfated maltopentaose, and sulfated maltohexaose.
  • a fifth aspect of the invention is the description of sulfated maltooligosaccharide compositions that differentially affect cell proliferation depending on the degree of sulfation of the compositions.
  • a sixth aspect of the invention is the description of highly sulfated maltooligosaccharide compositions that can be beneficially applied to the treatment or prevention of certain diseases including cancer, cardiovascular disease, retinopathies, inflammation, and diseases of viral origin.
  • Figure 1 shows the effects of the degree of sulfation of certain maltooligosaccharides on binding of RO-12 UC cells to bFGF coated microtiter wells.
  • Figure 2 shows the effects of the degree of sulfation of certain maltooligosaccharides on the inhibition of adrenocortical endothelial cell proliferation.
  • Figure 3 shows the inhibition of binding of RO-12 UC cells to bFGF coated microtiter wells as a function of the size of certain sulfated maltooligosaccharides.
  • Figure 4 shows the inhibition of adrenocortical endothelial cell proliferation as a function of the size of certain sulfated maltooligosaccharides.
  • Figure 5 shows the effects of highly sulfated maltotetraose on the growth of the tumor cell line MDA-231 in nude mice.
  • Figure 6 shows the effects of highly sulfated maltotetraose on CAPAN-2 tumor cell growth in nude mice.
  • Figure 7 shows the effects of highly sulfated maltotetraose on the growth of the tumor cell line, PC-3, in nude mice.
  • Figure 8 shows the inhibitory effects of highly sulfated maltotetraose on smooth muscle cell growth. The effects are depicted relative to porcine mucosal heparin.
  • Figure 9 shows the inhibitory effects of highly sulfated maltopentaose on smooth muscle cell growth. The effects are depicted relative to porcine mucosal heparin.
  • Figure 10 shows the inhibitory effects of highly sulfated maltohexaose on smooth muscle cell growth. The effects are depicted relative to porcine mucosal heparin.
  • Figure 11 shows the effects of highly sulfated maltotetraose on heparanase inhibition.
  • compositions having therapeutic properties similar to heparin are highly sulfated maltooligosaccharides having the general structure given by the formula:
  • each R 1 independently represents an alkyl, aryl or aralkyl group, a reduced or oxidized glucose unit, S0 3 M or H;
  • R 2 represents a S0 3 M group or H; M represents a biologically acceptable cation; and n represents an integer from 1 to 9; with the proviso that at least 50% of R 2 are S0 3 M.
  • Typical examples of biologically acceptable cations are alkali metals, alkaline earth metals, aluminum, ammonia, zinc, and substituted ammonia wherein the substitution may produce a di- or trialkylamine (C 1 -C 6 ), piperidine, pyrazone, alkanolamine (C 2 -C 6 ), or cycloalkylamine (C 3 -C 6 ), although acceptable cations are not limited to these. Any cation providing reasonable solubility and low or no toxicity and which does not significantly, or adversely affect the pharmaceutical properties of the parent composition is acceptable.
  • compositions of the instant invention are maltotetraose, maltopentaose, maltohexaose, maltoheptaose, maltooctaose, maltononaose and maltodecaose.
  • the compositions of the invention can be made by treating a maltooligosaccharide, or a derivative thereof as described below, with a sulfating agent in an appropriate solvent by methods which are well known in the art.
  • Maltooligosaccharide starting materials for the synthesis of the compositions of the instant invention include oligosaccharides having 1 to 9 D-glucose residues linked ⁇ -1 ,4 or mixtures of these oligosaccharides.
  • oligosaccharides such as O- or S-glycosides or reduced alditol derivatives
  • O- or S-glycosides or reduced alditol derivatives are useful as starting material as well. It will be apparent that the structure of the final sulfated oligosaccharide desired will determine the nature of the starting material.
  • Sulfating agents useful in preparing the compositions of the instant invention include but are not limited to sulfur trioxide:pyridine complex, sulfur trioxide:trimethylamine complex, and chlorosulfonic acid.
  • Organic solvents useful for the preparation of the compositions of the instant invention include but are not limited to N,N-dimethylformamide (DMF), dimethylsulfoxide, and pyridine.
  • DMF N,N-dimethylformamide
  • DMF N,N-dimethylformamide
  • sulfation of the hydroxyl groups can be obtained.
  • the sulfate groups can be modified to possess biologically acceptable cations, including but not limited to Na, K, Li, Ca, Mg, NH 4 , Al, ethanolamine, triethanolamine, morpholine, pyridine and piperidine.
  • a typical sulfation reaction is carried out by dissolving the starting material (0.5 g) in N,N-dimethylformamide (20 mL) and adding sulfur trioxide pyridine complex (2 eguivalents/OH groups). The mixture is stirred at room temperature for an appropriate time, preferably 2 days. The pH is adjusted to 9 by the addition of 1M NaOH, then the crude product is either precipitated by the addition of an organic solvent (as e.g. ethanol), or if this is not appropriate the mixture is evaporated to dryness under reduced pressure. The crude product is then purified to remove inorganic salts, and finally converted into the desired salt form.
  • An important aspect of the instant invention is the sulfate content of the products.
  • the products are characterized as the sulfur content determined by elementary analysis. Because of the hygroscopic nature of the sulfated compositions this cannot be regarded as a reliable method, therefore in the present work the sulfate content was determined from the carbon/sulfur ratio which is independent from the moisture content of the samples.
  • the sulfate content of the products can be expressed as the sulfation ratio, which is the ratio of the number of sulfated hydroxyl groups to the total number of hydroxyl groups in the starting material, expressed as percentage.
  • the sulfate esters prepared will vary in the number and position of sulfonic acid substituents. In most cases, a mixture of sulfate esters of the starting oligosaccharide(s) will be obtained. Preferred compositions have about 50% of the hydroxyl groups sulfated. More preferred are compositions having 75% or greater of the hydroxyl groups sulfated. Mixtures of different maltooligosaccharides may be sulfated and utilized for medical applications. For example, a maltooligomer mixture is commercially available which includes linear homologs from the tetrasaccharide to the decasaccharide. Such a mixture was highly sulfated using the conditions described herein (0.25g maltooligomer mixture in 10ml DMF treated with sulfur trioxide pyridine complex).
  • compositions of the invention can be provided with fluorescent, radioisotope, or enzyme labels as desired.
  • Conventional techniques for coupling of label to carbohydrates or related moieties can be used. Such techniques are well established in the art. See, for example, U.S. Patent No. 4,613,665.
  • the labeled mixtures of the invention may be used to identify sites of disease as well as in competitive immunoassays, and as a means to trace the pharmacokinetics of the compositions in vivo.
  • Suitable radioisotope labels for this purpose include hydrogen 3 , iodine 131 , indium 111 , technetium", phosphorus 32 , and sulphate 35 .
  • Suitable enzymic labels include alkaline phosphatase, glucose-6-phosphate-dehydrogenase, and horseradish peroxidase. Particularly preferred fluorescent labels include fluorescein and dansyl. A wide variety of labels of all three types is known in the art.
  • compositions of the instant invention are useful in medical applications for treating or preventing a variety of diseases including cancer, preferably metastatic cancer, inflammation, and diseases caused or exacerbated by platelet aggregation, or angiogenesis, and for the treatment of conditions or diseases which are characterized by excessive and destructive smooth muscle cell proliferation.
  • the instant compositions because of their angiostatic activity, will be preferably applied for the beneficial treatment of angiogenic-based diseases.
  • One such class of diseases is cancer or retinopathies.
  • a member of this latter class is diabetic retinopathy that will be favorably treated by the compositions of the instant invention.
  • Administration of the compositions of the invention is typically by routes appropriate for glycosaminoglycan compositions, and generally includes systemic administration, such as by injection.
  • Typical implants contain biodegradable materials such as collagen, polylactate, polylactate/polyglycoside mixtures, and the like. These may be formulated as patches or beads.
  • Typical dosages are in the range of 0.1 - 100 mg/kg/day on a constant basis over a period of 5-30 days. A particularly preferred dosage is about 0.3 mg/kg/hr, or, for a 70 kg adult, 21 mg/hr or about 500 mg/day.
  • the doses and periods of treatment will be chosen by the physician that best fit the need of the patient.
  • Injection subcutaneously at a lower dose or administered orally at a slightly higher dose than intravenous injection, or by transmembrane or transdermal or other topical administration for localized injury may also be effective.
  • Localized administration through a continuous release device, such as a supporting matrix, perhaps included in a vascular graft material, is particularly useful where the location of the trauma is accessible.
  • Formulations suitable for the foregoing modes of administration are known in the art, and a suitable compendium of formulations is found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA, latest edition.
  • compositions of the invention may also be labeled using typical methods such as radiolabeling, fluorescent labeling, chromophores or enzymes, and used to assay the amount of such compositions in a biological sample following its administration.
  • Suitable protocols for competitive assays of analytes in biological samples are well known in the art, and generally involve treatment of the sample, in admixture with the labeled competitor, with a specific binding partner which is reactive with the analyte such as, typically, an immunoglobulin or fragment thereof.
  • the antibodies prepared according to the invention, as described below, are useful for this purpose.
  • the binding of analyte and competitor to the antibody can be measured by removing the bound complex and assaying either the complex or the supernatant for the label. The separation can be made more facile by preliminary conjugation of the specific binding partner to a solid support.
  • Such technigues are well known in the art, and the protocols available for such competitive assays are too numerous and too well known to be set forth in detail here.
  • the oligosaccharide compositions of the invention are useful in therapeutic applications for the treatment of conditions or diseases which are characterized by excessive and destructive smooth muscle cell proliferation. These conditions freguently occur where the subject has been exposed to trauma, such as in the case of surgical patients. The trauma caused by wounds or surgery results in vascular damage and secondary smooth muscle cell proliferation, which results in vascular restenosis.
  • This undesirable result can occur after vascular graft surgery, heart transplantation, balloon or laser angioplasty, arterial traumatic injury, postsurgical repair of muscular arteries, long-term in-dwelling of arterial catheters, invasive arterial diagnostic procedures, kidney, lung or liver transplants, coronary artery bypass surgery, carotid artery bypass surgery, femoral popliteal bypass surgery, and intracranial arterial bypass surgery.
  • compositions of the instant invention are useful as inhibitors of smooth muscle cell proliferation, a preferred application is the inhibition of the proliferation of smooth muscle cells in blood vessel walls that occurs in response to vascular injury, and in association with certain disease states (Austin, G.E., et al., J Am Coll Cardiol (1985) .6:369-375).
  • NAC-antiproliferative heparin refers to a mixture of non-fragmented glycosaminoglycan chains obtained by treating commercially available heparin with periodate as described herein, which mixture substantially lacks anticoagulant activity but inhibits the proliferation of smooth muscle cells.
  • sulfated maltooligosaccharides exhibit significant anti-angiogenic or angiostatic activity.
  • Angiogenesis is the process whereby new blood vessels are produced. It is a process that may be associated with certain diseases, including arthritis, retinopathies, and the growth and metastasis of tumors. See, Mitchell and Wilks, Annual Reports in Medicinal Chemistry (Academic Press 1992) 27:139-148; Chapter 15.
  • compositions that stimulate or inhibit angiogenesis can be identified using several assays known in the art.
  • the chick chorioallantoic membrane (CAM) assay is commonly used.
  • CAM chick chorioallantoic membrane
  • certain heparinoids inhibit angiogenesis when administered with certain steroids. Folkman and Ingber, Ann. Surg. (1987) 206:374, Folkman et al., Science (1983) 221 :719.
  • compositions described herein also exhibit anti-angiogenic activity in the CAM assay, and thus can be used to treat angiogenic based diseases.
  • one such disease is cancer.
  • the anti-angiogenic activity of the highly sulfated maltooligosaccharides would account for, at least in part, their anti- cancer activity.
  • the metastatic spread of tumor cells throughout the body is thought to be facilitated by enzymes secreted by tumor cells that degrade components of the basement membrane, thereby allowing tumor cells to disseminate via the circulation.
  • One such enzyme is endo- ⁇ -D-glucuronidase, or heparanase, which degrades heparan sulfate glycosaminoglycans. Heparan sulfate is a prominent component of parenchymal cell basement membranes.
  • the highly sulfated maltooligosaccharides of the instant invention exhibits significant heparanase inhibitory activity as revealed using standard assays.
  • cancer, and other diseases that have as one element unwanted heparanase activity can be beneficially treated with the compositions of the invention.
  • Basic fibroblast growth factor is a small, heparin-binding polypeptide growth factor which is mitogenic for a variety of cell types of meso- and neuroectodermal origin.
  • the mitogenic activity of bFGF is believed to derive from its specific interaction with one or more high affinity transmembrane receptors in the tyrosine kinase gene family.
  • Basic FGF is also known to interact with cell surface and extracellular matrix heparan sulfate proteoglycan (HSPG), and such molecules are often referred to as "low affinity" receptors.
  • HSPG heparan sulfate proteoglycan
  • the protein also binds heparin quite strongly in vitro, and advantage of this is taken in the routine purification of bFGF on affinity columns of immobilized heparin. More recently, several studies have provided evidence that heparin or the heparan sulfate (HS) chains of HSPG may in fact be a cofactor that promotes or enhances the binding of bFGF to its high affinity receptors.
  • the bFGF binding properties of certain heparins or heparin like molecules are described by Ishihara, M., et al., Anal Biochem (1992) 202:310-315.
  • compositions to bind to bFGF and inhibit bFGF dependent cell growth can be shown using certain assays.
  • Assays for measuring the effect of heparinoids on bFGF are known in the art.
  • a cell based competitive binding assay is described by Ishihara, M., et al., Anal Biochem (1992) 202:310- 315.
  • the assay is based on the observation that bFGF binds to a lymphoblastoid cell line, transfected with hamster syndecan (RO-12 UC cells), and that this interaction can be inhibited by compositions that bind to bFGF.
  • compositions are that the inhibition of bFGF- dependent cell growth is dependent on the degree of sulfation of the maltooligosaccharide compositions, the highly sulfated compositions described herein being the most effective.
  • Heparin is known to have an anti-thrombotic effect, and at least in part this is a result of heparin's capacity to inhibit platelet aggregation. Interference with platelet aggregation causes a significant bleeding liability in some patients.
  • Certain NAC heparins exhibit both non-anticoagulant activity and inhibit platelet aggregation. See, for example, co-owned U.S. Patent Application, Serial No. 753,299, filed September 3, 1991 , or PCT Patent Application No. US92/02516, filed March 27, 1992.
  • the preferred compositions similar to heparin, of the instant invention inhibit platelet aggregation.
  • the average degree of sulfation of the products was deduced from the data of elementary analysis. Because of the hygroscopic nature of the products the carbon/sulfur ratio was used instead of the sulfur content.
  • compositions 1 , 2 and 3 were made in three parallel experiments. To a solution of maltohexaose (0.198 g) in N,N-dimethylformamide (10 mL) sulfur trioxide pyridine complex (0.191 g (Experiment 1), 0.382 g (Experiment 2), and 0.573g (Experiment 3), respectively) was added. The mixtures were stirred at room temperature for two days. The pH was adjusted to 9 with 1 M NaOH, and the mixtures were evaporated to dryness under reduced pressure.
  • compositions 1 , 2 and 3 were produced in Experiments 1 , 2 and 3, respectively.
  • Composition 4, highly sulfated maltohexaose was made as follows:
  • compositions 1 , 2, 3 and 4 The effect of Compositions 1 , 2, 3 and 4 on the binding of RO-12 UC cells to bFGF coated micro-titer wells was determined as described by Ishihara, M., et al., Anal Biochem (1992) 202:310-315. Bound cells are readily quantitated as total protein. Heparin which inhibits RO-12 UC cell binding was run as a positive control. The assay was run as follows: Fifty microliters of 10 ug/ml human recombinant bFGF was added to wells of a 96-well tissue culture plate and incubated overnight at 4°C.
  • RO-12 UC cells were suspended at a density of 3 x 10 6 cells/ml in PBS containing 5% fetal bovine serum. To this mixture was added the desired amount of sulfated composition, or heparin. They were made up in PBS plus 2.5% fetal bovine serum. A control was also run, containing only PBS plus 2.5% fetal bovine serum.
  • a second experiment was conducted.
  • the capacity of the sulfated maltohexaoses to inhibit the proliferation of a bFGF-dependent adrenocortical endothelial (ACE) cell line was determined.
  • This cell line (provided by D. Gospodarowicz, UCSF) requires either aFGF or bFGF for a proliferative response.
  • Cells were seeded at low density in microtiter wells in the presence of 2 ng/ml bFGF, and growth was determined as total protein after four days in the presence of the sulfated maltohexaoses. The results are shown in Figure 2. Again, the more highly sulfated maltohexaose compositions exhibited the highest anti-proliferative activities.
  • Example 2 Synthesis of Sulfated Maltotetraose and Biological Activity Maltotetraose may be purchased commercially from Sigma Corporation or can be produced enzymatically using the procedure of Ratanakhanockchal et al. See, Applied and Environmental Microbiology: vol. 58, no. 8, pages 2490-2494. Maltotetraose is sulfated as follows.
  • composition 5 The degree of sulfation of the product, denoted Composition 5, is shown in Table 2.
  • Example 4 Synthesis of Highly Sulfated Maltose and Maltotriose and Biological Activities
  • Maltose and maltotriose were sulfated and tested in the assays described above.
  • Maltotriose may be purchased commercially or can be produced enzymatically using the procedure of Ratanakhanockchal et al. See Applied and Environmental Microbiology:58, no. 8, pages 2490-2494.
  • Maltotriose was sulfated as follows. Maltotriose and maltose were sulfated using essentially the reaction conditions described for maltotetraose. In the case of maltose, after adjusting the pH to 9 the product was not collected by precipitation with ethanol, but by concentrating the reaction mixture under reduced pressure. The degree of substitution of the Composition 8 is shown in Table 4.
  • the mammary adenocarcinoma MDA231
  • the pancreatic tumor cell line CAPAN-2
  • the prostatic adenocarcinoma cell line PC-3. All cell lines are available from the American Type Culture Collection, and grow aggressively in nude mice. The experiments were conducted as follows: female, 15-20 gram nude mice, in groups of ten, were inoculated subcutaneously with 3-5 x 10 6 viable MDA231 , CAPAN-2, or PC-3 cells in 0.1 ml PBS. The cells were grown in standard DMEM tissue culture media supplemented with 10% fetal calf serum in a humidified 5% C02 incubator at 37°C. The cells were harvested with trypsin-EDTA, washed 2x with PBS, resuspended at a concentration of 3-5 x 10 7 cells/ml, and placed on ice prior to injection.
  • mice On a daily basis, the mice were subcutaneously injected with 100 mg/kg of highly sulfated maltotetraose made up in PBS, and produced as described in Example 2.
  • the composition was filter sterilized with a 0.2um Gelman filter unit. Tumor volume was determined using the following formula:
  • Tumor Volume Length x Width 2
  • mice were injected with PBS vehicle only.
  • Experimental and control mice were injected twice daily with 0.05 ml of the appropriate solution starting on days 0-40 post tumor challenge. From days 41-70 the animals were dosed once daily with 100mg/kg/day in a volume of 0.1 ml per injection, and tumor volume measured at defined times using standard methods.
  • PC-3 human prostatic adenocarcinoma line
  • This cell line is also available from the American Type Culture Collection. The materials and methods described above were similarly used here with the following exceptions.
  • the composition was administered subcutaneously in a single dose of 80 mg/kg/ day in a volume of 0.05 ml per dose Animals were dosed beginning 24 hours after tumor challenge. 5 x 10 6 PC-3 cells were injected in a volume of 0.1 ml subcutaneously in the anterior dorsal region (12 animals/treatment group).
  • Example 6 Inhibition of Smooth Muscle Cell Growth Maltotetraose, maltopentaose and maltohexaose were sulfated as described in Examples 1-3, and assayed for their effects on smooth muscle cell proliferation using a standard assay for this activity.
  • a convenient assay is as follows: Solutions to be tested are made up in DMEM medium containing 10% fetal calf serum and penicillin/streptomycin.
  • Bovine smooth muscle cells SMC are isolated from bovine aorta by the method of Ross, R., J Cell Biol (1971) 172-186. SMC from passage 3-10 are plated at 350-700 cells per well in 96-well microtiter plates in the medium above and allowed to attach for 2-4 hr. The complete medium is then replaced with DMEM supplemented with 0.1% fetal calf serum, and the cells are incubated for an additional period of about 24 to 72 hr to arrest cell growth. The low-serum medium is then replaced with complete medium containing the test samples.
  • the sulfated maltose oligosaccharides of the invention were added to the medium to make final concentrations as shown in the figures.
  • the effects of heparin at the same concentrations were also determined.
  • the results are plotted as the per cent of heparin inhibition where the concentration of heparin that gave 100% inhibition of SMC proliferation was 150 ug/ml.
  • the cells are allowed to grow for up to 7 days with replicate plates sampled at regular intervals.
  • Cell number is determined by removing the medium and washing the cells with phosphate-buffered saline, adding 75-150 ul lysis buffer, and assaying for lactate dehydrogenase (LDH) activity, as described by Brandley, B., et al., J Biol Chem (1987) 262:6431.
  • LDH lactate dehydrogenase
  • Figure 8 shows the anti-proliferative activity of highly sulfated maltotetraose. This composition exhibits significant anti- proliferative effects, although not as great as heparin. With respect to heparin, about 50% inhibition is observed at about 60 ug/ml.
  • Figure 9 shows the anti-proliferative activity of highly sulfated maltopentaose. This composition is more effective in inhibiting SMC proliferation than the smaller- sized sulfated maltotetraose. Indeed, 100% inhibition of SMC growth is observed at about 50 ug/ml. In contrast, one hundred per cent inhibition was not observed for highly sulfated maltotetraose at a concentration of 150 ug/ml.
  • Figure 10 shows the anti-proliferative activity of highly sulfated maltohexaose. This composition shows similar efficacy in comparison to highly sulfated maltopentaose. These date show that highly sulfated maltooligosaccharides of the invention are inhibitors of SMC growth, and thus, can be beneficially applied for the treatment of diseases where SMC growth is sought to be controlled or eliminated.
  • Compositions that stimulate or inhibit angiogenesis can be identified using several assays known in the art. Highly sulfated maltotetraose, Composition 5, Table 2, was tested for angiogenesis activity using the chicken chorioallantoic membrane (CAM) assay. The assay was performed as described by Castellot et. al., J. of Cellular Physiology (1986) 127: 323-329, with the exception that samples were evaluated for their efficacy to inhibit neovascularization. Carboxy methyl cellulose pellets containing 50 ug of hydrocortisone, or hydrocortisone plus different amounts of highly sulfated maltotetraose were incubated on the CAM for 3-4 days before scoring the results.
  • CAM chicken chorioallantoic membrane
  • Angiostatic activity is defined as a partial clearing or an avascular zone around the pellet.
  • pellets at each maltooligosaccharide concentration contained 50 ug of hydrocortisone.
  • the number in the Table is the number of embryos scored that exhibited no effect, a partial clearing (+), or an avascular zone (++).
  • Table 6 shows the results. It is apparent that the sulfated composition exhibits angiostatic activity. At low concentrations (eg. 3 and 6 ug/mL), the composition is slightly better at inhibiting the formation of new blood vessels than the buffer control, or hydrocortisone alone. At the higher concentrations, 12.5, 25, and 50 ug/ml, increase in partial clearing areas and avascular zone are observed. Table 6
  • Pellets 0.5% aqueous methylcellulose
  • maltoheptaose and maltose were tested for heparanase inhibitory activity using heparanase from a rat hepatoma cell line.
  • the cell line is described by Gerschenson, et al., Science (1970) 170: 859-861. Further, their inhibitory activities were compared to porcine mucosal heparin.
  • protease inhibitors were present in the MES buffer: 0.2 ug/ml aprotinin, 0.5 ug/ml leupeptin,100 ug/ml soybean trypsin inhibitor, 1 mM PMSF, 2 mM EDTA (sodium salt), and 15mM D-saccharic acid 1 ,4 lactone (exoglucuronidase inhibitor).
  • the cells were added to a 7ml Dounce homogenizer, freezed/thawed 3 times in an ethanol/dry ice bath, and homogenized with 15 strokes using a tight pestle.
  • the resulting cell lysates were placed in a 2 ml centrifuge tube and centrifuged at 4 ° C for 30 minutes at 16,000 x g. The supernatant was removed, and the protein concentration in the supernatant determined using the Macro BCA protein assay. BSA was used as a standard.
  • Heparanase activity was quantified by measuring soluble N- 3 H-acetylated pancreas heparan sulfate fragments derived from uncleaved N- 3 H-acetylated pancreas heparan sulfate distinguishable by cetylpyridinium chloride (CPC) precipitation.
  • N- 3 H-acetylated pancreas heparan sulfate had a weight average molecular weight, or Mw, of about 13,000. The following procedures were used.
  • rat hepatoma cell supernatant isolated as described above, containing 7.5 - 10 ug of protein in 50mM MES buffer, pH 5.2, containing 0.14M NaCI, 6mM sodium azide and the protease inhibitors described above, is added to siliconized 1.5 ml microcentrifuge tubes. Three to six replicates were run for each concentration of highly sulfated maltotetraose while three replicates were run for each concentration of porcine mucosal heparin. Controls were run to account for background counts. It was previously shown that the highest concentration of inhibitor does not affect precipitation of the intact radiolabeled heparan sulfate substrate.
  • the enzyme substrate inhibitor mixture was mixed, after which the tubes were incubated at 37°C for 20 minutes. After 20 minutes, the reaction was stopped by adding to the reaction tubes 150 ul of an aqueous heparin solution (0.33 mg/ml). 200 ul of 100mM sodium acetate pH 5.5 and 100 ul of CPC (0.6% in water) were then added. Three replicates maintained on ice were run as background controls (0 min.) in which the enzyme was added to the tubes immediately followed by heparin to terminate the reaction. The samples were processed as described for the 20 minute time points. Next, the tubes were vortexed, incubated for 60 minutes at room temperature, and then centrifuged for 10 minutes at 4,000 x g in a 5415C Eppendorf centrifuge. The supernatant was removed and assayed for 3 H by liquid scintillation counting.
  • Values represented are the mean differences between soluble CPM measured at 20 minutes at different heparin or highly sulfated maltotetraose, maltoheptaose, and maltose concentrations and soluble CPM measured at 0 minutes at 0 ug/ml +/- the standard deviation of the sum of the 20 and 0 minute variances.
  • Figure 11 shows that highly sulfated maltotetraose is approximately one half as effective as porcine mucosal heparin in inhibiting heparanase activity.
  • the IC 50 values of highly sulfated maltotetraose and porcine mucosal heparin were 9.0 and 4.1 ⁇ g/ml, respectively.
  • maltoheptaose sulfate was as active as heparin in the assay, whereas highly sulfated maltose had no activity at greater than 200 ug/ml.
  • Example 9 Svnergistic Effect of Highly Sulfated Maltooligosaccharides and Chemotherapeutic Drugs in the Treatment of Cancer
  • one mechanism whereby the highly sulfated maltooligosaccharides exert their anticancer effect is by blocking angiogenesis.
  • experiments would be done to show a synergistic effect of these compositions with chemotherapeutic drugs that are known to be effective for treating cancer.
  • mice would be subcutaneously injected with 100 mg/kg of highly sulfated maltotetraose, Composition 5, Table 2, made up in PBS, produced as described in Example 2, with methotrexate. Methotrexate would be used at a concentration of 4 mg/kg.
  • the composition would be filter sterilized with a 0.2um Gelman filter unit. Similar to Example 5 tumor volume would be determined using the following formula:
  • Tumor Volume Length x Width 2
  • mice Three groups of control mice would be run. They would be injected with either PBS vehicle, 100 mg/kg of highly sulfated maltotetraose in PBS, or
  • mice 4 mg/kg methotrexate in PBS.
  • Experimental (highly sulfated maltotetraose with methotrexate) and control mice would be injected twice daily with 0.05 ml of the appropriate solution starting on days 0-40 post tumor challenge. From days 41-70 the animals would be dosed once daily with 100mg/kg/day in a volume of 0.1 ml per injection, and tumor volume measured at defined times using standard methods.
  • mice treated with the combination of highly sulfated maltotetraose and methotrexate would present an average tumor volume less than that observed with either agent alone. Tumor volume would be less than 60mm 3 at about days 68-69.

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Abstract

L'invention concerne une nouvelle catégorie de maltooligosaccharides fortement sulfatées présentant des propriétés semblables à l'héparine. L'invention traite également de procédés permettant d'utiliser ces oligosaccharides pour traiter certaines maladies dont le cancer, les rétinopathies, et les maladies cardiovasculaires. La figure illustre les effets du degré de sulfatage de certains maltooligosaccharides, sur la liaison d'une lignée cellulaire lymphoblastoïde transfectée avec des cellules (RO-12 UC) de syndécan d'hamster avec des cupules de microtitrage recouvertes d'un facteur de croissance de fibroblastes basique.
EP94931314A 1993-10-07 1994-10-04 Maltooligosaccharides fortement sulfatees presentant des proprietes semblables a l'heparine Withdrawn EP0722326A4 (fr)

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US133483 1993-10-07
PCT/US1994/011368 WO1995009637A1 (fr) 1993-10-07 1994-10-04 Maltooligosaccharides fortement sulfatees presentant des proprietes semblables a l'heparine

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AUPN261895A0 (en) * 1995-04-28 1995-05-18 Australian National University, The Preparation and use of sulfated oligosaccharides
AUPO556297A0 (en) * 1997-03-11 1997-04-10 Australian National University, The Sulfated oligosaccharides having anticoagulant/ antithrombotic activity
AUPO888497A0 (en) * 1997-09-01 1997-09-25 Australian National University, The Use of sulfated oligosaccharides as inhibitors of cardiovascular disease
DE19747195A1 (de) * 1997-10-24 1999-04-29 Knoll Ag Verwendung von Glykosaminoglykanen zur Herstellung von pharmazeutischen Zubereitungen zur Behandlung von mit Diabetes assoziierten Augenkrankheiten
WO2000062785A1 (fr) * 1999-04-15 2000-10-26 Takara Shuzo Co., Ltd. Remedes
US6608042B2 (en) * 2000-03-28 2003-08-19 Aventis Pharma, S.A. Pharmaceutical compositions containing oligosaccharides, the novel oligosaccharides and preparation thereof
ES2431362T3 (es) 2001-09-12 2013-11-26 Sigma-Tau Research Switzerland S.A. Derivados de glicosaminoglicanos totalmente N-desulfatados como agentes inhibidores de la heparanasa, dotados con actividad antiangiogénica y desprovistos de efecto anticoagulante
CN103788141B (zh) * 2004-03-04 2016-08-17 普罗吉恩制药有限公司 硫酸化寡聚糖衍生物
CN100363373C (zh) * 2005-01-14 2008-01-23 庄茅 异麦芽低聚糖硫酸酯（imos）的制备方法
MX338213B (es) * 2007-10-16 2016-04-07 Progen Pharmaceuticals Ltd Derivados de oligosacaridos sulfatados novedosos.
CA2861077A1 (fr) * 2012-01-30 2013-08-08 Baxter International Inc. Polysaccharides sulfates ou sulfones non anticoagulants
EP2832848B1 (fr) 2012-03-30 2019-02-13 Ajinomoto Co., Inc. Milieu de culture pour faire proliférer une cellule souche, qui contient un composé sulfaté
US11787783B2 (en) 2016-12-13 2023-10-17 Beta Therapeutics Pty Ltd Heparanase inhibitors and use thereof
JP2020503377A (ja) 2016-12-13 2020-01-30 ベータ セラピューティクス プロプライアタリー リミティド ヘパラナーゼ阻害剤及びそれの使用

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EP0722326A4 (fr) 1999-10-06

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