Classifications

• • 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
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H17/00—Compounds containing heterocyclic radicals directly attached to hetero atoms of saccharide radicals
  • C07H17/04—Heterocyclic radicals containing only oxygen as ring hetero atoms
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H3/00—Compounds containing only hydrogen atoms and saccharide radicals having only carbon, hydrogen, and oxygen atoms
  • C07H3/06—Oligosaccharides, i.e. having three to five saccharide radicals attached to each other by glycosidic linkages
• • 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
  • C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
  • C08B37/0075—Heparin; Heparan sulfate; Derivatives thereof, e.g. heparosan; Purification or extraction methods thereof
  • C08B37/0078—Degradation products

Definitions

• the invention relates to the use of
• carbohydrate preparations as therapeutic and diagnostic compositions.
• the invention relates to polysaccharides having six or more saccharide units and compositions containing such polysaccharides which are useful in treating diseases and conditions characterized by excessive smooth muscle cell proliferation.
• N-acetyl-D-glucosamine GlcNAc
• D-glucosamine N-sulfate GlcNS
• 2,5-anhydromannose Man(2,5);
• 2,5-anhydromannitol ManH(2,5);
• D-xylose Xyl;
• glycosaminoglycan GAG.
• S O-linked sulfate residues
• alpha and beta anomeric linkages are as those conventionally found in heparin and the indicated D or L configurations as conventionally found pertains.
• D or L configurations as conventionally found pertains.
• Glycosaminoglycans are copolymers of alternating hexosamine and aldouronic acid residues which are found in sulfated forms and are synthesized as proteoglycans. They have collectively been called muco- polysaccharides, and those in heparin are more precisely called glycosaminoglycuronans.
• heparin and heparan sulfate are members of the GAG family which are classified by the nature of the hexosamine/aldouronic acid repeating units.
• the aldouronic acid is primarily D-glucuronic acid
• the hexosamine is N-acetylated 2-amino-2-deoxy-D-galactose, more commonly known as N-acetyl galactosamine and abbreviated as
• the aldouronic acid is mostly L-iduronic acid and the hexosamine is GalNAc.
• the aldouronic acid is replaced by D-galactose, and the hexosamine is mostly N-acetylated 2-amino-2-deoxy-D-glucose, more commonly known as N-acetyl glucosamine and abbreviated as GlcNAc.
• the hexosamine is mostly N-acetylated or N-sulfated glucosamine (GlcN), and the aldouronic acid is mostly L-iduronic in heparin and mostly D-glucuronic acid in heparan sulfate.
• GlcN N-acetylated or N-sulfated glucosamine
• the aldouronic acid is mostly L-iduronic in heparin and mostly D-glucuronic acid in heparan sulfate.
• Heparan sulfate is commonly considered to have a higher
• Conventional heparin (used as an anticoagulant) has a molecular weight of 5-25 kDa and is extracted as a mixture of various chain lengths by conventional procedures. These procedures involve autolysis and extraction of suitable tissues, such as beef or porcine lung, intestine, or liver, and removal of other GAGs as well as nonpolysaccharide components.
• the molecular weight of the chains in the extract is significantly lower than the 60-100 kd known to exist in the polysaccharide chains of the heparin proteoglycan synthesized in the tissue.
• the GAG moiety is synthesized bound to a peptide matrix at a serine residue through a tetrasaccharide linkage region of the sequence D-GlcA-D-Gal-D-Gal-D-Xyl ⁇ protein, which is then elongated at the D-GlcA residue with alternate additions of GlcNAc and GlcA.
• the polysaccharide sidechains are modified by a series of enzymes which sequentially deacetylate the N-acetyl glucosamine and replace the acetyl group with sulfate, epimerize the hydroxyl at C5 of the D-glucuronic acid residue (to convert it to L-iduronic acid), sulfate the 0-2 of the resulting L-iduronic acid and the 0-6 of the glucosamine residue.
• Some of the chains are further sulfated at the 0-3 of the glucosamine residue, either at the heparan or heparin stage. This latter sulfation generates the active sequence required for anti- thrombin III binding and thus anticoagulation activity.
• Other chemically possible sulfation sites are on the 0-2 of D-glucuronic acid.
• heparin may contain considerable amounts of what might otherwise be classified as heparan sulfate.
• Heparin and heparan sulfate can slow or arrest the vascular smooth muscle cell proliferation associated with injury
• glycosaminoglycans operate, or to what extent they interact with other growth factors such as epithelial and fibro-blast growth factors. It has been proposed that a 3-0 sulfate on glucosamine in an oligosaccharide of at least
• Tetrasaccharides of the type tested were shown to have very low antiproliferative activity; hexasaccharides, octasaccharides, and decasaccharides were shown to be active to approximately the same level on a weight/volume concentration basis. Also tested was a synthetic
• pentapeptide which represents a unique sequence of the heparin required for the binding of heparin to
• pentapeptide i.e., periodate treatment
• does not destroy antiproliferative activity i.e., periodate treatment
• polysaccharides containing 6 or more sugar residues which oligosaccharides have enhanced antiproliferative activity with respect to smooth muscle cells.
• the invention provides a low molecular weight glycosaminoglycan (GAG) composition which has superior specific antiproliferative activity with regard to smooth muscle cells.
• GAG glycosaminoglycan
• the existence of this activity in a low molecular weight GAG provides the opportunity for effective pharmaceutical compositions which can be prepared by synthesis or by isolation of the composition from natural sources.
• the invention is directed to a process to prepare a sulfated
• polysaccharide having antiproliferative activity having antiproliferative activity.
• the polysaccharides of the invention may be produced
• the polysaccharide compounds of the invention synthetically it is first necessary to synthesize an iduronic acid synthon. Next, a glucosamine synthon is produced. The iduronic acid synthon and glucosamine synthon are reacted to produce a disaccharide synthon. The disaccharide units can be reacted to form oligosaccharides containing 4, 6, 8 or any multiple thereof of monosaccharide units and/or can be reacted with either an iduronic or glucosamine reaction synthon to provide oligosaccharides containing any odd number of saccharide units.
• the heparin is obtained from a natural source and subjected to digestion with nitrous acid under conditions which favor the formation of an oligosaccharide mixture containing large amounts of hexa- and octasaccharides.
• the mixture is separated according to size and those factions corresponding to hexa- and octasaccharides are combined and recovered.
• the recovered portions are then separated according to charge in order to obtain the more highly charged fractions.
• oligosaccharides which are highly sulfated. Polysaccharides sulfated at the 0-3 position of the GlcN (associated with anticlotting activity) are not encompassed by the present invention.
• the invention is also directed to
• compositions comprised of the
• oligosaccharides of the invention either alone or in combination with excipients, i.e., pharmaceutically acceptable materials with no pharmacological effect.
• compositions may be administered to a patient in order to regulate smooth muscle cell proliferation.
• a primary object of the present invention is to provide synthetically produced oligosaccharides
• Another important object of the present invention is to provide a method of obtaining hexa- and octasaccharide units from natural heparin and heparan sulfate which hexa- and octasaccharide units are
• compositions which can be administered to aid in the regulation of smooth muscle cell
• oligosaccharide units include monosaccharide residues which are sulfated at particular positions (other than the 0-3 position) which effected the ability of the oligosaccharide to regulate smooth muscle cell
• Figures 1A and 1B show the elution profiles from gel filtration chromatography of reaction mixtures produced using varying amounts of nitrous acid.
• Figure 2 shows the growth inhibition activity of the various sized fractions.
• Figures 3A and 3B show the elution profiles of hexasaccharide and octasaccharide subunits, respectively, from DEAE-Toyopearl chromatography.
• Figures 4A and 4B show the growth inhibition activity of various fractions collected in the elution profiles of Figures 3A and 3B.
• Figure 5A shows the elution profile from reverse-phase ion-pairing HPLC for the S-6 fraction shown in Figure 3A.
• Figure 5B shows a comparable profile for the total hexasaccharide fraction.
• Figures 6A, 6B and 6C are charts showing possible sulfated positions for fifty-seven octasaccharides of the invention.
• heparin/heparan sulfate or "heparin” is meant a preparation obtained from tissues in a manner conventional for the preparation of heparin as an anticoagulant or otherwise synthesized and corresponding to that obtained from tissue. See Conrad, H.E., Heparin and Related Polysaccharides, Vol. 56, p. 18 of Annals of N.Y., Academy of Sc, June 7, 1989, incorporated herein by reference. This preparation may include residues of D-glucuronic acid (GlcA), as characteristic of heparan sulfate as well as iduronic acid (IdoA) as characteristic of heparin. However, both GlcA and IdoA are present in both, they are present in different proportional amounts. The (IdoA)/GlcA ratio increases as heparan sulfate becomes more heparin-like. As described in the
• heparin/heparan sulfate or "heparin” is intended to cover the range of mixtures encountered.
• heparin/heparan sulfate preparation can be obtained from a variety of rriammalian tissues
• heparin/heparan sulfate starting material including, if desired, human tissue.
• porcine or bovine sources are used, and vascularized tissues are preferred.
• a preferred source of heparin/heparan sulfate starting material is porcine intestinal mucosa, and preparations labeled "heparin" prepared from this tissue source are commercially available.
• the heparin/heparan sulfate starting material is prepared from the selected tissue source by allowing the tissue to undergo autolysis and extracting the tissue with alkali, followed by coagulation of the protein, and then
• the complex is recovered by reprecipitation with a polar nonaqueous solvent, such as ethanol or acetone or their mixtures, and the fats are removed by extraction with an organic solvent such as ethanol and proteins by treatment with a proteolytic enzyme, such as trypsin.
• a polar nonaqueous solvent such as ethanol or acetone or their mixtures
• an organic solvent such as ethanol and proteins
• proteolytic enzyme such as trypsin.
• Suitable procedures for the preparation of the heparin starting material are found, for example, in Charles, A.F., et al., Biochem J (1936) 30: 1927-1933, and modifications of this basic procedure are also known, such as those disclosed by Coyne, E., in Chemistry and Biology of Heparin. Elsevier Publishers, North Holland, New York, Lunblad, R.L., et al., eds.
• the synthetic oligosaccharides of the present invention include at least 6 saccharide residue units and have the following general structural formula:
• each of the variables A, B, C and D are independently hydrogen or SO 3 R with the proviso that at least 2 of the variables are SO 3 R and each R is
• R 1 and R 2 are each independently hydrogen, or one or more repeating units having the following structure:
• Some preferred embodiments of the present invention include compounds of structural Formula I wherein each of A, B, C and D are -SO- and either R 1 or R 2 is the unit of structural Formula 1(a).
• Another preferred embodiment includes compounds of structural Formula I wherein each of A, B, C and D are -SO- and either R 1 or R 2 is the unit of structural Formula 1(a).
• Another preferred embodiment includes compounds of structural Formula I wherein each of A, B, C and D are -SO- and either R 1 or R 2 is the unit of structural Formula 1(a).
• Another preferred embodiment includes compounds of structural Formula I wherein each of A, B, C and D are -SO- and either R 1 or R 2 is the unit of structural Formula 1(a).
• the heparin/heparan sulfate preparation used as a starting material is first purified by extraction with a solvent in which the heparin is insoluble, such as ethanol or acetone.
• a solvent in which the heparin is insoluble such as ethanol or acetone.
• the purified start- ing material is then depolymerized.
• Depolymerization in general can use various reagents, such as nitrous acid, heparinase or periodate.
• reagents such as nitrous acid, heparinase or periodate.
• the antiproliferative compositions of the invention are obtainable when partial nitrous acid digestion is
• the nitrous acid is prepared in situ by acidification of a solution of sodium nitrite at a concentration of 50 mM, and the reagent is used to treat the heparin at a concentration of about 60-180 mg/ml, at a pH of about 1.0 to about 2.0, preferably about 1.5.
• the reaction is conducted at room temperature and can be neutralized by addition of a suitable reagent at the desired stage of digestion.
• depolymerization methods can be used as long as they produce active components, i.e. components which (1) are predominantly hexa- and octasaccharides; (2) are heavily sulfated; (3) have substantial antiproliferation activity with respect to smooth muscle cells; and (4) have insignificant or no anticlotting activity.
• Isolated fragments can then be tested for their ability to inhibit smooth muscle cell proliferation.
• the depolymerization results in a mixture of fragments that is then separated on the basis of size.
• size separation techniques including gel permeation, density gradient
• centrifugation especially preferred is gel filtration chromatography using a Sephadex or polyacrylamide gel system with a fractionation range of about 100-3500 daltons.
• a particularly preferred gel permeation resin is Biogel P10, and upon separation using this method, fragments which are disaccharides, tetrasaccharides, hexasaccharides, octasaccharides, and oligosaccharides of higher molecular weights are effectively separated.
• fractions containing predominantly hexa- and octasaccharide units show enhanced activity in inhibiting the proliferation of smooth muscle cells.
• Verification of this property can be obtained using standard assays, such as those described in Castellot, J.J. Jr., et al., J Cell Biol (1986) 102:1979-1984.
• each of the variables A, B, C and D is independently H or SO 3 R and each R is independently H or a cation, with the proviso that at least two of the
• variables A, B, C or D is -SO 3 R. It is pointed out that hydroxyl groups on 3-positions of the sugars have been omitted for greater clarity and the * adjacent the COOH indicates undetermined stereochemistry.
• the cations represented by R can either be inorganic cations such as sodium, potassium, calcium, or ammonium ion or can be organic cations such as those obtained from quaternary amines; these salts are formed by simple neutralization.
• each "R" can be any cation
• the above eleven possible structures represent a significantly larger number of compounds, i.e., the acid and salt forms.
• L-iduronic acid IdoA
• D-glucosamine GlcNH 2
• N-acetyl-D-glucosamine GlcNAc
• D-glucosamine N-sulfate GlcNS
• 2,5-anhydromannose Man(2,5)
• 2,5-anhydromannitol ManH(2,5).
• S The location of the O-linked sulfate residues is indicated by "S” and the number of the position of sulfation where the SO 3 R residue is linked to oxygen.
• alpha and beta anomeric linkages are as those shown in formula 1 above and the indicated D or L configurations as set forth above pertains.
• the locations of the sulfates are shown below the abbreviation for the sugar to which they apply.
• n 1 or 2
• each of the variables A, B, C and D is independently H or SO 3 R, wherein each R is
• the cations represented by R can either be inorganic cations such as sodium, potassium, calcium, or ammonium ion or can be organic cations such as those obtained from quaternary amines and these salts are formed by simple neutralization.
• the hydroxyls at the 3 positions are not shown in the structure, but are understood to be present, and the asterisk adjacent the position of the carboxyl groups indicates that the stereochemistry at these positions is undetermined.
• formula (III) it can be seen that there are fifty-seven different possible configurations with respect to the position of the -SO 3 R moieties when 2 or more are present.
• each "R" can be H or a cation
• the fifty-seven possible structures represent a significantly larger number of compounds, i.e., the acid and salt forms.
• Preferred compounds of the invention are the hexasaccharides.
• the preferred octasaccharides include octasaccharides having antiproliferative activity with smooth muscle cells which have the formula
• octasaccharides wherein at least two IdoA-GlcNY units are
• preferred octasaccharides of the invention include any of the following octasaccharides, their pharmaceutically acceptable salts and mixtures of two or more of such octasaccharides and their salts
• the oligosaccharide compositions of the invention are useful in therapeutic applications for treatment of conditions or diseases which are characterized by excessive and destructive smooth muscle cell proliferation. These conditions frequently 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 secondary proliferation results in vascular resenosis.
• 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 intra- cranial arterial bypass surgery.
• compositions of the invention are useful in treatment.
• Administration is by typical routes appropriate for polysaccharide compositions, and generally includes systemic administration, such as by injection.
• Particularly preferred is intravenous injection, as continuous injection over long time periods can be easily continued.
• Typical dosage ranges are in the range of 0.1-10 mg/kg/hr on a constant basis over a period of 5-30, preferably 7-14, days.
• Particularly preferred dosage is about 0.3 mg/kg/hr, or, for a 70 kg adult, 21 mg/hr or 540 mg/day.
• compositions of the invention may also be labeled using typical methods such as radiolabeling, fluorescent labeling, chromophores or enzymes, and used in a competitive assay for the amount of
• analyte such as, typically, an immunoglobulin or fragment thereof.
• the antibodies prepared according to the invention 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
• the antibodies of the invention are useful in immunoassays, not only of the type described above involving competition between labeled composition and the analyte antiproliferation factor in the sample, but also for direct immunoassay for the factor. Alternate
• protocols involving direct assays are also of wide variety and well known.
• the analyte bound to antibody is detected by means of an additional reactive partner which bears a label or other means of detection.
• the binding of the antibodies of the invention to analyte can be detected by further reaction with a labeled
• the antibodies of the invention can also be formulated into pharmaceutical compositions and used to stimulate the growth of smooth muscle cells in subjects for which this result is desirable.
• BioGel chromatography two columns were connected in tandem, each approximately 5 cm in diameter, 128 cm in length, were packed with a total of 5 1. of BioGel P10. The columns were prepared and run in 0.5 M NH 4 HCO 3 at a flow rate of 0.7 ml per min.
• Bovine smooth muscle cells were isolated from bovine pulmonary artery by the method of Ross, R.J., Cell Biol (1971) 172-186. SMC from passage 3-10 were 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 was replaced with DMEM supplemented with 0.1% fetal calf serum, and the cells were incubated for an additional period of about 24 to 72 hr to arrest cell growth. The low-serum medium was then replaced with complete medium containing the test samples.
• the cells were allowed to grow for up to 7 days with replicate plates sampled at regular intervals. Cell number was determined by removing the medium and washing the cells with phosphate-buffered saline, adding
• LDH dehydrogenase
• the DEAE-toyopearl chromatography was run on a larger scale using a 5 cm x 27 cm column packed in 0.3 M NH 4 HCO 3 . Up to 3 g of oligosaccharide mixture was loaded onto this column and the column was washed successively with 2 1 volumes of 0.3 M, 0.5 M, .06 M, 0.9 M, and 1.2 M NH 4 HCO 3 . The fraction emerging in 0.9 M NH 4 HCO 3 ,
• Figure 5A shows the elution profile from the highest charged fragment of DEAE-Toyopearl
• Figure 5B shows the results using the total hexasaccharide
• the charge separated fraction is a greatly simplified mixture.
• the individual components of this simplified mixture are expected to have antiproliferative activity.
• the more highly charged fragments generally show (as compared with less highly charged fragments and/or commercial heparin) (1) a greater ability to inhibit the proliferation of smooth muscle cells and (2) a lesser ability to act as an anticoagulant.
• fractionation/separation processing can be carried out which improve factors (1) and (2) and also simultaneously aid in eliminating fragments which include
• Preferred oligosaccharide fragments of the invention possess characteristic (1) and (2) and (3) are highly charged and (4) include a very low (or no) amounts of saccharides sulfated at the 3-position as compared with fragments of commercial heparin. In order to obtain such preferred oligosaccharides, it is preferable to produce them synthetically rather than obtain them from digestion of heparin.
• the oligosaccharides of the invention which are particularly preferred have a number of distinct characteristics such as greater ability to inhibit proliferation of smooth muscle cells, lesser ability to act an as anticoagulant, high degrees of sulfation, and lack of sulfation at the 3-position.
• Oligosaccharides regardless of their size or whether their glucosamine unit is sulfated at 0-6, can be retrosynthesized to a common protected disaccharide unit.
• This disaccharide unit is open for chain extension in both directions; its thioglycoside function permits chain-extension towards the reducing end, whereas
• chloracetyl group allows further chain-extensions towards the non-reducing end.
• the amino groups of the target compounds are masked as azido groups, which assure advantageous stereocontrol in glycosylation reactions.
• Benzyl (Bn) groups are used as permanent blocking groups for the hydroxyls which are nonsulfated in the target compounds, and the semi-permanent benzoyl (Bz) group is used for the OH's to be sulfated.
• the p-methoxybenzyl (MBn) group stands for hydroxyl groups the sulfation of which is optional in the target compounds.
• the disaccharide synthon should be available by glycosylating the protected 2-azido-2-deoxy-glucopyranosyl derivative with the iduronosyl bromide.
• This disaccharide can be synthesized by coupling the same iduronic acid donor with a glucosamine derivative shown in brackets.
• protecting group with the p-methoxybenzyl group allows specific deprotection and subsequent sulfation in any order, leading to structures which have sulfate groups in positions: a) masked by benzoyl groups in the protected derivative; b) masked by p--ethoxybenzyl groups in the protected derivative; and c) masked by both benzoyl and p-methoxybenzyl groups in the protected derivative.
• a further advantage of the use of the p-methoxybenzyl group is that if selective removal of this group is not required it can be removed by catalytic hydrogenation in the same step as the permanent benzyl groups, thereby reducing the number of required synthetic steps.
• the glucosamine synthon for the reducing end was synthesized by an analogous sequence from methyl 2- benzyloxycarbonylamino-2-deoxy- ⁇ -D-glucopyranoside
• iduronosyl bromide was performed using silver triflate, in combination with collidine as a buffer of the reaction medium, and resulted in the disaccharides.
• the two disaccharide synthons were coupled by using dimethyl (methylthio) sulfonium triflate (F ⁇ gedi, P. et al., Carbohydr Res (1986) 149:C9. incorporated herein by reference) (DMTST) to give the tetrasaccharide with the required ⁇ -interglycosidic linkage.
• DMTST dimethyl (methylthio) sulfonium triflate
• Patent 4,943,630 issued July 24, 1990, which patent is incorporated herein by reference to disclose methods of synthesizing oligosaccharides. While the present invention has been described with reference to specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalence may be submitted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit and scope of the invention. All such

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