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WO1994008595A1 - Utilisation d'heparine non anticoagulante dans le traitement des lesions ischemiques et consecutives a une perfusion - Google Patents

Utilisation d'heparine non anticoagulante dans le traitement des lesions ischemiques et consecutives a une perfusion Download PDF

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Publication number
WO1994008595A1
WO1994008595A1 PCT/US1993/009794 US9309794W WO9408595A1 WO 1994008595 A1 WO1994008595 A1 WO 1994008595A1 US 9309794 W US9309794 W US 9309794W WO 9408595 A1 WO9408595 A1 WO 9408595A1
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heparin
anticoagulant
ischemia
reperfusion
nac
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PCT/US1993/009794
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English (en)
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Michael Sobel
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Virginia Commonwealth University
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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/70Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters

Definitions

  • This invention is in the field of cardiovascular diseases. More specifically, it is directed to beneficial applications of essentially non- anticoagulant heparin oligomers for treating or preventing ischemia/reperfusion tissue injury in an animal.
  • D-glucuronic acid GlcA
  • L-iduronic acid IdoA
  • D-glucosamine • GlcNH2
  • N-acetyl-D-glucosamine GlcNAc
  • D-glucosamine N- sulfate GlcNS
  • 2,5-anhydromannose AMan
  • 2,5-anhydromannitol AManH.
  • each saccharide residue below the appropriate abbreviation, the location of the 0-linked sulfate residues is indicated by "S" and the number of the position of sulfation where the sulfate residue is linked to oxygen on the sugar residue.
  • S the number of the position of sulfation where the sulfate residue is linked to oxygen on the sugar residue.
  • the positions involved in the alpha and beta anomeric linkages are as those conventionally found in heparin, ⁇ . (glucosamine ⁇ uronic) and ⁇ (uronic ⁇ glucosamine) , and the D or L configurations as conventionally found pertains.
  • the locations of the sulfates are shown below the abbreviation for the sugar to which they apply, thus, for example, IdoA-GlcNS
  • 2S _ 6S refers to a dimer of L-iduronic acid and D-glucosamine N-sulfate-linked ⁇ (l-4) with sulfates connected respectively at the 2 and 6 positions of the sugar residues.
  • Ischemia/Reperfusion Injury Tissue injury caused by ischemia and reperfusion is a common surgical problem.
  • the ischemia- reperfusion syndrome contributes significantly to increased morbidity, limb loss, and death.
  • the most effective therapy is fasciotomy.
  • Other therapies include intravenous mannitol infusion which may reduce the generation of toxic oxygen free radicals that occur and damage tissue during reperfusion. McCord, J.M. New England Journal of Medicine (1985) 312:159-163.
  • Other free radical scavengers have also been used including superoxide dismutase.
  • heparin may be beneficial and reduce muscle necrosis caused by the ischemia/reperfusion syndrome. Hobson, R. . et al. Journal of Vase. Surg. (1988) 7:585-591. However, heparin's effectiveness in treating ischemia-reperfusion is counter balanced by potential increases in bleeding associated with its administration. Wright, J.G. et al. Archives of Surgery (1988) 123:470-472.
  • Heparin/heparan sulfate is a member of a class known as glycosaminoglycans (GAG) . These materials are copolymers of alternating hexosamine and aldouronic acid residues which are found in sulfated forms and are synthesized as proteoglycans.
  • GAG glycosaminoglycans
  • the hexosamine is mostly N-acetylated or N- sulfated glucosamine (GlcNAc and GlcNS)
  • 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 proportion of glucuronic acid than heparin. Problems of heterogeneity in preparations of heparan sulfate or heparin isolated from tissues make sharp distinctions difficult.
  • Conventional heparin (used as an anticoagulant) has a molecular weight of 5-25 kd 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 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.
  • heparin may contain considerable amounts of what might otherwise be classified as heparan sulfate.
  • Non-anticoagulant Heparin There is a body of art that describes the production of non-anticoagulant heparin. Most of the publications describe non-anticoagulant heparin produced from depolymerized heparin/heparan sulfate, and separation of products by size. In a generally used procedure, the heparin starting material is depolymerized in the presence of nitrous acid with or without pretreatment to remove acylation from any GlcNAc residues present. Nitrous acid, under the appropriate conditions, cleaves at the linkage between a GlcNS or GlcNH 2 residue and the uronic acid residue through which it is linked through a glucosamine c-(l-4) uronic acid linkage.
  • the heparin has been deacetylated, all of the glucosamine - uronic acid residues are susceptible and complete depolymerization results in disaccharides. If the heparin has not been deacetylated, the glucosamine ⁇ uronic acid residues wherein the glucosamine is acetylated are resistant, and both disaccharides and tetrasaccharides containing the resistant linkage result. In all cases, the glucosamine residue at the reducing terminus of the disaccharide or tetrasaccharide is converted to a 2,5-anhydromannose in the course of cleavage.
  • IdoA-GlcNS 2S wherein the glucosamine residue is sulfated at the 2 and/or 6 position in an arbitrary manner, and wherein some of the IdoA residues may be replaced by cleaved IdoA 2S or GlcA residues resulting from the periodate oxidation.
  • These shortened polymeric chains are said to lack the binding site for ATIII but to be capable of inhibiting smooth muscle proliferation and to have physiological activities that include acceleration of tissue repair, prevention of atherogenous lesions, prevention of states of shock, and prevention of the development of metastasis.
  • the invention is directed to a method of treating or preventing ischemia/reperfusion tissue injury in an animal host consisting of administering an effective amount of a non- anticoagulant heparin.
  • a second aspect of the invention consists of a method of treating or preventing ischemia/reperfusion tissue injury in an animal host by administering an effective amount of a non-anticoagulant heparin, preferably via intravenous or oral administration.
  • a third aspect of the invention is directed to a method of treating or preventing ischemia/reperfusion tissue injury in an animal host consisting of. administering an effective amount of a substantially non-fragmented non-anticoagulant heparin.
  • a fourth aspect of the invention is directed to a method of treating or preventing ischemia/reperfusion tissue injury in an animal host consisting of administering an effective amount of a non-anticoagulant heparin which is produced from heparin by oxidation with periodate and subsequent reduction.
  • Figures 1A and IB show the time' course of periodate oxidation of heparin under two conditions: pH 3, 4°C and pH 6.5, 37°C.
  • Figures 2A-2D show the effect of periodate oxidation on various moieties in heparin at pH 3, 0°C.
  • Figure 3 shows the experimental design for perfusing non-anticoagulant heparin to ischemic rat limbs. Arrows below the time line indicate measurement of flow rates which were subsequently used to calculate the total vascular resistance of the hindlimb. Phenylephrine was added directly to the perfusate, and acetylcholine (0.001, 0.01, 0.1. 1.0 ug/min) and nitroprusside (0.5, 5.0, 50 ug/min) were delivered by constant infusion into the arterial cannula. With the exception of the 60 minute ischemic interval, non-ischemic and ischemia/reperfusion groups were treated identically.
  • Figure 4 compares the per cent change in total vascular resistance as a function of various concentrations of acetylcholine for post-ischemic or control animals treated with or without non- anticoagulant heparin or dextran sulfate.
  • the invention relates to methods for treating ischemia/reperfusion injury in an animal host with a non-anticoagulant heparin.
  • a non-anticoagulant heparin is substantially non-fragmented and prepared from commercially available heparin. More preferred the non-anticoagulant heparin is prepared by oxidization with periodate and reduction by sodium borohydride. Described herein are methods to prepare these compositions and the nature of the resulting composition.
  • non-anticoagulant heparins described by Fransson, L.-A. and Lewis, W. , FEBS Lett (1979) .97:119-123, or in U.S. Patent No. 4,990,502, or by Casu, B. et al. , Arzneim Forsch/Drug Res (1986) 16:637-642 may be used with the methods of the instant 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
  • 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.
  • GlcA D-glucuronic acid
  • IdoA iduronic acid
  • both GlcA and IdoA are present in both, they are present in different proportional amounts.
  • the (IdoA) /GlcA ratio rises as heparan sulfate becomes more heparin-like.
  • heparin/heparan sulfate or "heparin” preparation can be obtained from a variety of mammalian tissues, including, if desired, human tissue. Generally, porcine or bovine sources are used, and vascularized tissues are preferred. A preferred source of heparin starting material is porcine intestinal mucosa, and preparations labeled "heparin" prepared from this tissue source are commercially available.
  • the heparin 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 precipitation of the heparin-protein complex from the supernatant by acidification.
  • 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
  • a 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) 10:1927-1933, and modifications of this basic procedure are also known, such as those disclosed by Coyne,
  • NAC-heparin refers to a mixture of substantially non-anticoagulant fragmented or n ⁇ n- fragmented heparin obtained by subjecting commercially available heparin to one or more chemical treatments. Where stated in the specification, it refers to periodate oxidized heparin as described herein, which mixture substantially lacks anticoagulant activity and inhibits the proliferation of smooth muscle cells.
  • the invention method uses NAC-antiproliferative heparin to treat or prevent tissue injury associated with ischemia/reperfusion.
  • the preferred NAC- antiproliferative heparin is described in co-owned U.S. Patent Application Serial No. 753,299, filed September 3, 1991.
  • An additional preparation is described in co-owned U.S. Patent Application Serial No. 677,737, filed March 29, 1991.
  • the heparin starting material is treated with periodate under conditions wherein the diols on adjacent carbons contained in the glycosaminoglycan structure are oxidized to the corresponding aldehydes.
  • Any glucuronic acid or iduronic acid which does not contain either or both a 2-sulfate or 3-sulfate would therefore be "susceptible" to oxidation and cleavage.
  • the susceptible iduronic acid residues are cleaved much more rapidly than the susceptible glucuronic acid residues.
  • susceptible glucuronic acid residues which are conjugated to the reducing terminus of a GlcNS residue are much less rapidly cleaved and oxidized than those susceptible glucuronic acid residues which are linked to the reducing terminus of a GlcNAc residue.
  • the glucuronic acid residues which reside in the typical ATIII binding saccharide sequence are preferentially oxidized, and by suitable adjustment of the time course of oxidation, a large proportion of the ATIII binding activity can be destroyed without affecting in a substantial way, the antiproliferative activity.
  • Any free amino groups on glucosamine residues will also be oxidized; however, the majority of glucosamine residues in heparin are either sulfated or acetylated. These residues, therefore, are not affected by the periodate oxidation.
  • NAC nonanticoagulant
  • periodate oxidation is performed in 0.01-0.10 M sodium periodate buffered to a pH of 3- 6, preferably with 0.05-0.2 M sodium acetate or sodium phosphate buffer.
  • Reaction mixtures containing commercially-available heparin/heparan sulfate at 0.5-10% (wt./volume) are incubated with the periodate oxidation solution at 0-37°C in dark amber containers for time intervals generally greater than 3 hrs. While this temperature range is workable, lower temperatures are greatly preferred, especially in the range of 0°-5°C, especially 0°- 1°C.
  • the concentration of the oxidized heparin in the reduction mixture is 1-20% (w/v) .
  • Excess borohydride is then destroyed by addition of concentrated HC1 to approximately pH 4. The pH is then readjusted to neutrality with 2 M sodium carbonate and the product is desalted and dried.
  • the resulting composition contains modified but unfragmented heparin/heparan sulfate of molecular weight in the range of 5-25 kd with an average chain length of 50-100 saccharide units.
  • the composition is a mixture of oxidation products corresponding to the original mixture of glycosaminoglycans in the heparin preparation, but is free of other biological contaminants.
  • the composition is useful therapeutically under circumstances where antiproliferative activity is desirable.
  • the anticoagulant activity of the original heparin/heparan sulfate preparation is reduced to less than 40 u/mg, preferably less than 5 U/mg, as opposed to 170 U/mg in the original preparation.
  • the inhibition of smooth muscle cells by the preparation is the same as or greater than that of the original heparin on a weight basis.
  • the heparin starting material is subjected to two distinct chemical reactions to convert it to a non-anticoagulant heparin.
  • the method consists of first N-deacetylating N-acetylhexosamine, followed by a second reaction which consists of oxidizing deacetylated heparin, preferably with periodate.
  • the reaction consist of dissolving heparin (Ming Han heparin, 170Umg) in distilled water to give 540 ml of a solution containing 80 g heparin. Eighteen grams of hydrazine sulfate are dissolved in 1260 ml of anhydrous hydrazine in a 3 liter beaker and the heparin solution added to the beaker with stirring. The reaction is allowed to proceed for 6 hours at about 98°C, and the deacetylated product is isolated.
  • deacetylated heparin is next oxidized in solution containing 0.5-10% deacetylated heparin (w/v) with 0.01-0.10 M periodate at pH3-6 at 0°-37° for a time sufficient to affect complete oxidation of heparin by periodate.
  • the aldehyde groups produced are reduced to alcohols by treatment with sodium borohydride at about 0.1-0.3 M, pH 8.9.
  • Ischemia-Reperfusion Injury A number of animal models systems may be utilized to show the efficacy of NAC heparin for treating or preventing ischemia-reperfusion tissue injury.
  • a canine skeletal muscle ischemia/reperfusion model is described by Hobson et al. in Journal of Vase. Surg. (1988) 7:585-590.
  • a rat hindlimb ischemia/reperfusion model is described by Wright et al. Journal Cardiovasc. Surg. (1991) 32 (for supplement) :11.
  • An additional rat model system is described by Sternberg and Edelman, Journal Vase. Surg. (1992) 16:30-39.
  • NAC heparin for treating or preventing ischemia-reperfusion tissue injury in the instant invention was demonstrated using an isolated rat hindlimb model.
  • This model system as well as the others mentioned above, are correlative and predicative of the efficacy of NAC heparins in animals including humans. Briefly, the procedure consists of perfusing isolated rat hindlimbs with an appropriate physiological solution and causing ischemia by halting perfusate flow. A contralateral limb serves as a control which is subjected to constant perfusate flow. See Sternberg et al. Journal of Vase. Surg. (1992) 16:30-39.
  • NAC-heparin treated limb and the control limb are treated identically except the former is subjected to sixty (60) minutes of ischemia, and is perfused with pre- and post-ischemic perfusate containing NAC-heparin.
  • NAC heparins To determine the beneficial effects of NAC heparins on ischemia/reperfusion injury, its effects on both endothelial cell- dependent and independent vasoreactivity is determined by measuring the change in vascular resistance in response to increasing amounts of acetylcholine and nitroprusside, respectively, infused into the artery.
  • Intact vasculature of an organ must be preconstricted to facilitate measurement of vasodilator responses to agonists.
  • the base line was established by measuring native venous flow rates of non-ischemic or ischemic limbs by perfusing the limbs with Kreb's buffer containing phenylephrine for 10 minutes to pre-constrict the vasculature. This pre- constricted flow rate was then recorded which was used as the base line for subsequent measurements.
  • Total vascular resistance may be calculated by dividing the perfusion pressure by the flow rate and expressed as mm Hg/ml/min/g dry limb weight. The results can be expressed as the percent reduction in endothelial-dependent vasal dilation to acetylcholine when compared to the non-ischemic controls.
  • rat hindlimbs in both the experimental and control groups are perfused with
  • NAC-heparin is included in the perfusate experimental group which is subsequently subjected to sixty (60) minutes of ischemia. Next, reperfusion is conducted for ten (10) minutes with NAC heparin. Endothelial cell function is assessed in both the experimental and control groups as a function of total vascular resistance in response to various concentrations of acetylcholine. Endothelial cell independent vasoreactivity is assessed with nitroprusside.
  • Non-Anticoagulant compositions of the invention can be provided with fluorescent, radioisotope, or enzyme labels as desired.
  • the labeled mixtures of the invention may be used to identify sites of ischemia 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", and phosphorus 32 .
  • 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.
  • non-anticoagulant heparin compositions of the invention are useful in therapeutic applications for treating or preventing tissue injury caused by ischemia/reperfusion.
  • Administration is by typical routes appropriate for glycosaminoglycan 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. Also preferred are introduction into the vascular system through ' intraluminal administration or by adventitial administration using osmotic pumps or implants.
  • Typical implants contain biodegradable materials such as collagen, polylactate, polylactate/polyglycoside mixtures, and the like. These may be formulated as patches or beads.
  • 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 about 500 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 antiproliferative component in a biological sample.
  • 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 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 techniques 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 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 preparation of these same antibodies or by labeled antibody immunoreaetive with this preparation by virtue of species differences.
  • 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.
  • porcine mucosa heparin (Ming Han heparin, 900201, 170 u/mg) was dissolved in 450 ml of distilled water and 50 ml of 1 M sodium acetate buffer, pH 5.2, was added. The solution was transferred to a 1 1 amber bottle and chilled to 0°C in an ice bath. After the temperature had equilibrated 500 ml of prechilled 0.2 M sodium periodate was added with moderate stirring.
  • the solution of oxidized heparin was filtered through Whatman #1 paper to remove a small amount of flocculent precipitate and the filtrate was dialyzed against four changes of distilled water (vol ratio 1:10) using a 3.5 kDa cutoff membrane.
  • the volume was then reduced to 400 ml by ultrafiltration using a Pharmacia Tangential Flow Apparatus fitted with a Nova 1 kDa molecular weight cut-off membrane.
  • the concentrated solution was chilled to 0°C in an ice-water bath and 8.3 g of NaHC0 3 was added.
  • a solution containing 3.78 g NaBH 4 in 100 ml of 0.05 M Na 2 C0 3 pre-chilled to 0°C was added to the reaction mixture and the reduction was allowed to proceed at 0°C with moderate stirring.
  • the pH of the reaction mixture was 8.5 at the beginning of the reaction and rose to 9.5 as the reaction proceeded. After 2 hr the pH was adjusted to 4.0 by addition of 6 N HCl and the mixture was allowed to stand for 30 min at room temperature to destroy the excess NaBH 4 . Finally, the pH was adjusted to 7.0.
  • the solution containing the final product was dialyzed as described above and then lyophilized to dryness.
  • the overall yield was 90% of the starting weight of heparin.
  • the product was dissolved in distilled water to give a 5% solution (wt/vol) and reprecipitated with 3 volumes of 99% ethanol.
  • the precipitate was washed three times with 99% ethanol and the remaining ethanol was removed by placing the powder in the lyophilizer for 1 hr.
  • porcine heparin (Ming Han Batch HM900201) was periodate oxidized at a concentration of 0.8% of heparin in a reagent solution of 20 mM NaIO , 20 mM NaH 2 P0 ⁇ ,, 20 mM Na 2 HP0 , and 0.2 M NaCl (pH 6.5).
  • the reaction mixture was prepared by addition of 1500 ml of 1.6% heparin (25 grams) in deionized water to 1500 ml of 40 mM NaI0 4 , 40 mM NaH 2 PO ⁇ ,-40 mM Na 2 HP0 4 , 0.4 M NaCl with moderate stirring at room temperature.
  • the mixture was transferred to three 1 liter brown bottles immediately after the mixing.
  • the reaction was run at 37°C for 24 hrs in an incubator. After the reaction, excess periodate was consumed by adding 16 ml of ethylene glycol (to give a final concentration of 100 mM) to the reaction mixture and incubating at 37°C for 1 hr.
  • reaction mixture was then concentrated to 475 ml by ultrafiltration using a Pharmacia
  • the pellets were dissolved in deionized water and dialyzed against water for 48 hrs in a 1000 Da cut-off dialysis tube with three changes of water.
  • the dialyzed material was lyophilized to dryness to generate the nonanticoagulant heparin (7 grams) as the retentate.
  • the solutions outside of the dialysis tube were combined, concentrated, dialyzed and lyophilized to generate lyophilized dialysate (4 grams) .
  • Example 2 Properties of the NAC-antiproliferative Compositions
  • the NAC-antiproliferative heparin prepared according to paragraph A of Example 1 shows less than 5 u/mg anticoagulant activity compared to 170 u/mg for the starting material.
  • NAC-antiproliferative heparin preparation in paragraphs B and C were tested using intravenous delivery in the assay described hereinabove using 29 male Sprague Dawley FBR albino rats weighing approximately 350 grams.
  • the animals in groups 1 and 2 received the NAC-antiproliferative heparin prepared as in paragraphs B and C, respectively, at the rate of 0.3 mg/kg/hr for 14 days in lactated Ringer's solution.
  • Group 3 received lactated Ringer's solution alone.
  • NAC-antiproliferative heparin was as effective in preventing myointimal hyperplasia as heparin. It was also observed that no visible bleeding occurred post surgery in any of the rats, and the animals took less time to recover and looked healthier than those previously treated with whole heparin. Additional studies on the NAC-antiproliferative preparation as set forth in paragraphs B and C of paragraphs 1 showed almost exclusively the presence of long oligomers with only a few shorter chains. Earlier preparations which resulted in significant depolymerization gave less satisfactory results.
  • NAC-antiproliferative heparins prepared in Example 1 were also analyzed for disaccharide composition by complete hydrolysis in the presence of nitrous acid, as described in Guo, Y. , and Conrad, H.E., Anal Biochem (1989) 176:96-104.
  • Hydrolysis with nitrous acid cleaves at N-sulfated glucosamine residues (but not at N-acylated glucosamine residue) and converts the reducing terminus to 2,5- anhydromannose.
  • Subsequent reduction of this residue to 2,5-anhydromannitol is used to stabilize the cleavage products in this assay.
  • the various hydrolysis products are quantitated relative to
  • IdoA-AManH 2S 6S which is known not to be destroyed in periodate oxidation and is set at 100.
  • NAC- antiproliferative composition A comparison of the composition of the NAC- antiproliferative composition with that of heparin is shown in Table 1. "ND" represents not detectable. As shown in Table 1, disaccharide segments susceptible to periodate oxidation (i.e., those containing unsulfated IdoA or GlcA) are completely destroyed. Those units expected not to be susceptible to periodate oxidation (those containing 2S IdoA or 2S GlcA) are retained at approximately the same ratio to the resistant standard as their occurrence in heparin.
  • the disaccharide and tetrasaccharide compositions obtainable by degradation of the original heparin and of the oxidized samples were measured to follow the destruction of the uronic acid residues of the heparin during the oxidization.
  • Each sample was treated with nitrous acid at pH 1.5 and the resulting di- and trisaccharides were quantified using the reversed phase ion pairing HPLC method described previously (Guo, Y. and Conrad, H.E. Analyt Biochem (1989) 176:96-104) .
  • Anticoagulant activity was determined by APTT and anti-Xa assays. These assays were kindly performed by Dr. Betty Yan, Lilly Research Labs, Indianapolis, IN, USA.
  • the trisulfated disaccharide that is critical for the anticoagulant activity is the trisulfated disaccharide that is critical for the anticoagulant activity.
  • GMS 2 3S,6S abbreviated here as GMS 2 .
  • Direct nitrous acid cleavage of this segment of heparin yields the tetrasaccharide, tl4, and not free GMS 2 .
  • GMS 2 in heparin is situated in a position with a GlcNS residue linked to the C4 position of its GlcA, it will be released by nitrous acid without prior N-deacetylation as the free disaccharide.
  • Other possible degradation products and their abbreviations are:
  • ISMS IdoA-AMan
  • IMS IdoA-AMan
  • 6S and GMS GlcA-AMan 6S.
  • Figure 1 shows a comparison of the rates of disappearance of the major disaccharide units of heparin at pH's 3.0 (4°C) ( Figure la) and 6.5 (37°C) ( Figure lb) .
  • the samples at various time intervals were N-deacetylated and then cleaved with nitrous acid at both pH 1.5 and pH 4.0 to give total disaccharide release.
  • the disappearance of each susceptible disaccharide is due to the oxidation of its uronic acid residue. Only those uronic acid residues that lack a SO A substituent at both C2 and C3are susceptible to IO A " .
  • Venous flow from the hindlimb was measured by timed collection over a 30 second interval, normalized to ml/min, and expressed per gram dry limb weight (ml/min/g dry limb weight) . Venous effluent was not recirculated. It is to be noted that because this ischemia/reperfusion injury model has a constant perfusion pressure, the total vascular resistance of the hindlimb can be calculated directly from the flow rate. All limbs were initially perfused for a twenty limbs.
  • Hindlimbs that were subjected to ischemia/reperfusion were also subjected to an equivalent period of 20 minutes stabilization perfusion followed by sixty (60) minutes of total ischemia at 37°C.
  • the limbs were reperfused for 10 minutes with buffer containing phenylephrine. Thereafter, vasoreactivity to acetylcholine and nitroprusside were tested as performed in the non- ischemic limbs.
  • both the non-ischemic and ischemia/reperfusion hindlimbs were subjected to identical amounts of perfusion prior to administration of the agonists.
  • the drug was added to the pre-and post-ischemic perfusate.
  • acetylcholine was used at four concentrations: 0.001, 0.01, 0.1, and 1.0 ⁇ gms/min.
  • nitroprusside was used at three concentrations: 0.5, 5.0, and 50 ⁇ gms/min.
  • NAC heparin was used at a concentration of 4 ⁇ gms/ml.
  • Acetylcholine and nitroprusside were suspended in normal saline and delivered into the arterial perfusate line with an infusion pump. Flow rates became steady state after 10 minutes of perfusion with phenylephrine and 1-1.5 minutes with acetylcholine and nitroprusside.
  • Total vascular resistance was calculated by dividing the perfusion pressure (70 mm Hg) by the flow rate and expressed as mmHg/ml/min/g dry limb weight. See, Sexton, W.L., et al. Journal of Applied Phvsiol . (1990) 68.:387-392. The effect of NAC on TVR was expressed as a percent change from the phenylephrine-preconstricted base line. See, Mugge, A., et al . American Journal of Physio. (1991) 260-H242-H247. Data was analyzed using a 1-way analysis of variance with Tukey's studentized range test for comparison of variance between any two groups. Groups were considered significantly if p ⁇ 0.05. All data are presented as mean ⁇ S.E.

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  • Veterinary Medicine (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Polysaccharides And Polysaccharide Derivatives (AREA)

Abstract

Procédé de traitement ou de prévention des lésions ischémiques ou consécutives à une perfusion chez un mammifère, qui consiste à administrer audit mammifère, de préférence sous forme orale ou intraveineuse, une quantité efficace d'une héparine non anticoagulante.
PCT/US1993/009794 1992-10-13 1993-10-13 Utilisation d'heparine non anticoagulante dans le traitement des lesions ischemiques et consecutives a une perfusion WO1994008595A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU54425/94A AU5442594A (en) 1992-10-13 1993-10-13 Use of non-anticoagulant heparin for treating ischemia/reperfusion injury

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US95982392A 1992-10-13 1992-10-13
US07/959,823 1992-10-13

Publications (1)

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WO1994008595A1 true WO1994008595A1 (fr) 1994-04-28

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2803522A1 (fr) * 2000-01-06 2001-07-13 Aventis Pharma Sa Nouvelle application therapeutique de l'enoxaparine
EP1289508A2 (fr) * 2000-05-02 2003-03-12 CHARLOTTE-MECKLENBURG HOSPITAL doing business as Carolinas Medical Center Procede pour prevenir l'apoptose
US6787365B2 (en) * 1998-02-09 2004-09-07 The Regents Of The University Of California Inhibition of L-selectin and P-selectin mediated binding using heparin
WO2012146774A1 (fr) 2011-04-28 2012-11-01 Endotis Pharma Conjugués d'oligosaccharide dans la prévention de l'ischémie-reperfusion
WO2013095279A1 (fr) * 2011-12-19 2013-06-27 Dilafor Ab Nouveau glycosaminoglycan faiblement anticoagulant
US9480702B2 (en) 2011-12-19 2016-11-01 Dilaforette Ab Use of chemically modified heparin derivates in sickle cell disease
WO2022203571A1 (fr) * 2021-03-24 2022-09-29 Tx Medic Ab Combinaison pour le traitement d'une thromboinflammation

Citations (2)

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Publication number Priority date Publication date Assignee Title
WO1992017188A1 (fr) * 1991-03-29 1992-10-15 Glycomed, Inc. Nouveaux derives de l'heparine non anticoagulants
WO1992017187A1 (fr) * 1991-03-29 1992-10-15 Glycomed, Inc. Nouveaux derives de l'heparine non anti-coagulants

Patent Citations (2)

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WO1992017188A1 (fr) * 1991-03-29 1992-10-15 Glycomed, Inc. Nouveaux derives de l'heparine non anticoagulants
WO1992017187A1 (fr) * 1991-03-29 1992-10-15 Glycomed, Inc. Nouveaux derives de l'heparine non anti-coagulants

Non-Patent Citations (5)

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Title
HOBSON, R.W. ET AL: "ROLE OF HEPARIN IN REDUCING SKELETAL MUSCLE INFARCTION IN ISCHEMIA-REPERFUSION", MICROCIRCULATION, ENDOTHELIUM AND LYMPHATICS, vol. 5, no. 3-5, 1989, pages 259 - 276 *
PUKAC, L.A. ET AL: "ANTIPROLIFERATIVE EFFECTS OF NOVEL, ANTICOAGULANT HEPARIN DERIVATIVES ON VASCULAR SMOOTH MUSCLE CELLS IN VITRO AND IN VIVO", THE AMERICAN JOURNAL OF PATHOLOGY, vol. 139, no. 6, December 1991 (1991-12-01), pages 1501 - 1509 *
SOBEL, M. ET AL: "FLUORESCENT LABELING OF HEPARINS AND RELATED POLYSACCHARIDES", ACS SYMPOSIUM SERIES, POLYMERIC DRUGS AND DRUG DELIVERY SYSTEMS, vol. 469, 1991, pages 60 - 70 *
STERNBURGH, W.C. ET AL: "HEPARIN PREVENTS POSTISCHEMIC ENDOTHELIAL CELL DYSFUNCTION BY A MECHANISM INDEPENDENT OF ITS ANTICOAGULANT ACTIVITY", JOURNAL OF VASCULAR SURGERY, vol. 17, no. 2, February 1993 (1993-02-01), pages 318 - 327 *
WRIGHT, J. G. ET AL: "PRE-ISCHAEMIC HEPARINIZATION DECREASES RAT SKELETAL MUSCLE REPERFUSION INJURY", THE JOURNAL OF CARDIOVASCULAR SURGERY, vol. 32, no. SUP., 1991, pages 11 *

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6787365B2 (en) * 1998-02-09 2004-09-07 The Regents Of The University Of California Inhibition of L-selectin and P-selectin mediated binding using heparin
FR2803522A1 (fr) * 2000-01-06 2001-07-13 Aventis Pharma Sa Nouvelle application therapeutique de l'enoxaparine
WO2001049298A3 (fr) * 2000-01-06 2002-04-04 Aventis Pharma Sa Nouvelle application therapeutique de l"enoxaparine
EP1289508A2 (fr) * 2000-05-02 2003-03-12 CHARLOTTE-MECKLENBURG HOSPITAL doing business as Carolinas Medical Center Procede pour prevenir l'apoptose
JP2003531854A (ja) * 2000-05-02 2003-10-28 シャーロット‐メクレンバーグ・ホスピタル・オーソリティ,ドゥーイング・ビジネス・アズ・キャロライナズ・メディカル・センター アポトーシスの予防方法
WO2012146774A1 (fr) 2011-04-28 2012-11-01 Endotis Pharma Conjugués d'oligosaccharide dans la prévention de l'ischémie-reperfusion
WO2013095279A1 (fr) * 2011-12-19 2013-06-27 Dilafor Ab Nouveau glycosaminoglycan faiblement anticoagulant
US9475888B2 (en) 2011-12-19 2016-10-25 Dilafor Ab Non anti-coagulative glycosaminoglycans comprising repeating disaccharide unit and their medical use
US9480702B2 (en) 2011-12-19 2016-11-01 Dilaforette Ab Use of chemically modified heparin derivates in sickle cell disease
US9480701B2 (en) 2011-12-19 2016-11-01 Dilaforette Ab Low anticoagulant heparins
WO2022203571A1 (fr) * 2021-03-24 2022-09-29 Tx Medic Ab Combinaison pour le traitement d'une thromboinflammation

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