US 3766167 A
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Oct. 16, 1973 s. E. LASKER ETA].
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BY MARHE L. CHHJ m AZ M ATTORNEY United States Patent 01 ice 3,766,167 Patented Oct. 16, 1973 3,766,167 ORALLY ACTIVE ANTICOAGULANT Sigmund E. Lasker, New York, N.Y., and Marie L. Chiu, Edgewater, N.J., assignors to Research Corporation, New York, N.Y.
Filed Mar. 26, 1971, Ser. No. 128,447 Int. Cl. C07c 95/04 US. Cl. 260-211 R 1 Claim ABSTRACT OF THE DISCLOSURE Disclosure describes a new orally effective anticoagulant derived from heparin by controlled hydrolysis.
BACKGROUND OF THE INVENTION Anticoagulants are widely employed for the prevention or retardation of intravascular blood clotting in thrombophlebitis and in embolism or occlusion of peripheral pulmonary or coronary arteries in mammals.
The principal anticoagulants presently employed by veterinarians and physicians are heparin which is a naturally occurring product isolated from animal tissues and bishydroxy-coumarin, and chemical analogs and derivatives of this compound. Heparin is utilized by injection. The coumarin type compounds are orally efiective. The onset of heparin activity is rapid. The coumarin drugs require an induction period which may amount to several hours. They are, however, effective for relatively long periods of time. Standard methods of emergency treatment at the present time are to administer heparin by injection for its rapid anticoagulant effect and a coumarin orally for maintenance of the activity.
Because of the general aversion to injection techniques and dangers sometimes associated therewith, the art has long sought an orally effective anticoagulant which would reach therapeutic levels of activity rapidly.
The invention It has now been discovered that by controlled hydrolysis of the parenterall active anticoagulant heparin having a molecular weight of 15000 daltons it is possible to obtain on orally active anticoagulant having a molecular weight of 5300 daltons as determined by standard equilibrium ultracentrifugation techniques.
The orally active anticoagulants of this invention are polysaccharides. By elemental analysis the sodium salt as isolated contains about 22% carbon, 3.6% hydrogen, 2.5% nitrogen, 11.4% sulfur and 13.2% sodium. The molar ratio CzNzSzNa is about 10:1:223. It gives a positive metachromatic test for sulfated polysaccharides. It is completely soluble in water and insoluble in both acetone and ethanol.
The compound contains iduronic acid, glucuronic acid and glucosamine which may be cleaved by acid hydrolysis and separated.
The standard test for glucuronic and iduronic acids is generically defined as uronic acid test. The uronic acid content of the anticoagulating agent of this invention as determined by the carbazol method is 38.9% compared to 34.4% for heparin. As determined by the orcinol method the uronic acid content is 12.5% compared to 13.5% for heparin. The compound may be further distinguished from heparin by high voltage electrophoresis. It has been observed that at 1900 volts, 30 ma. pH 2 acetic-formic acid buifer, the compound migrates during a period of 90 minutes a distance which is 1.2 to 1.3 times the distance which the original heparin migrates.
The compound of the invention is prepared by enzymatic hydrolysis of heparin with heparinase. Heparinase is a known enzyme which can be isolated in accordance with the procedure described by Payza et al. in Biochem. Biophys. Acta. 20, 596 (1956). Hydrolysis takes place in an aqueous medium at a pH of from about 7.8 to 8.4 at a temperature of from about 20 C. to 35 C. during a period of from about 4 to 70 hours.
The preferred conditions from the point of view of yield and ease of operation and pH 8.1 to 8.3, temperature 25 C. to 30 0., time 48 to 70 hours.
The pH may be maintained in the desired range utilizing a butter. Specific samples of heparinase, in common with many enzymes will vary appreciably in their activity depending principally upon their method of isolation. A few simple observations will be sufficient to permit the skilled operator to determine optimum conditions for a particular heparinase.
The compound as isolated is in the form of a sodium salt. It may readily be converted to the free acid, to another metallic salt or to the acid addition salt. The compound contains a carboxyl group. Accordingly, acid addition salts will be prepared from acids which give a higher hydrogen ion concentration than does the carboxyl group. It presently appears that the sodium salt will be the anticoagulant of choice, but for man therapeutic uses the compound isolated will often be converted to parmacologically acceptable salts which as aforesaid may be either metallic salts or acid addition salts. Metallic salts may be prepared from alkali metal and alkaline earth metal bases preferably hydroxides. Acid addition salts may be prepared from acids such as hydrochloric, sulfuric, phosphoric, citric, and the like.
For the preparation of the acid salts it is convenient to simply titrate a water solution of the compound as isolated with a solution of the selected acid. Metallic salts may be prepared, for example, by passing a solution of the isolated sodium salt over a sulfonated polystyrene ion exchange resin on the acid cycle and treating the eluate with a dilute solution of the selected salt, suitably in the form of the hydroxide. In both instances the desired product can be obtained by freezedrying.
The products of this invention are useful as anticoagulating agents and for their antilipemic activity in mammals. The physician or veterinarian will determine a dosage which will be most suitable for a particular application. It may vary from patient to patient depending upon the size of the patient, the condition under treatment, and other circumstances, particularly level of prothrombin and other coagulating factors. All of these factors are readily evaluated by those skilled in the art. The products will normally be made available at a variety of dosage levels since, as is well known, different subjects, and even the same subject, will require different dosage levels for any of a number of reasons at the time of the administration of the therapeutic agent. For example, dosage units containing from 25 to 200 mg. may be useful.
The products of this invention may be administered alone but will generally be administered with a pharmaceutically acceptable non-toxic carrier the proportions of which are determined by the suitability and chemical nature of the particular carrier, the chosen rate of administration and standard pharmaceutical practice. For maintaining therapeutically effective levels in the blood they will normally be administered orally in the form of tablets or capsules containing such excipients as starch, sugar, certain types of clay, etc.
Although it is presently believed that the compounds of this invention will be most useful for oral administration they may be administered parenterally, for example, by intravenous or intramuscular injection. Dosage units may be prepared in the form of sterile water solutions containing other solutes, for example, enough saline or glucose to make the solution isotonic.
The following examples are given by way of illustration only and are not to be considered limitations of this invention, many apparent variations of which are possible without departing from the spirit or scope thereof.
EXAMPLE 1 Preparation of polysaccharide A total of 1.2 g. of heparin was inoculated with 600 mg. of heparinase prepared in accordance with the method described by Payza, A. N., Korn E. D., Biochem. Biophys. Acta. 20, 596 (1956), in 50 ml. of 0.02 N- phosphate buffer at pH 8.2 and held at C. for seventy hours. At the end of this period the reaction is stopped by the addition of cold 20% trichloroacetic acid. The mixture is filtered and filtrate is dialyzed for about sixteen hours against distilled water in seamless cellulose dialyzer tubing. The solution which remains in the tubing is lyophylized to obtain the desired product. The compound was characterized as a sodium salt.
Elemental analysis.-Carbon, 21.1%; nitrogen, 2.48%; hydrogen, 3.58%; sulfur, 11.39%; sodium, 13.25%. C:N:S:Na is 10:1:2z3. Molecular weight by ultra centrifuge: 5300 daltons soluble in water; insoluble in acetone and ethanol; anticoagulant activity in vitro; U.S.P. Method No. XVII is 70 I.U/mg. compared to 160 I.U./mg. for original heparin. The metachromatic test is positive.
EXAMPLE 2 Animal testing A chinchilla rabbit weighing 2.2 kg. having a normal control clotting time of 6 minutes by the Lee-White method was given mg. of the polysaccharide of Example 1 sublingually. The mouth was taped closed during the test period. A sample of cardiac blood was withdrawn 30 minutes after the sublingual application. The clotting time for the cardiac blood was 12 minutes. Subsequent blood samples were taken from an ear vein cannula. The clotting time remained elevated for two hours and subsequently returned to normal. The Lee- White method for coagulation time is described in Gradwohls Clinical Laboratory Methods and Diagnosis, vol. II, p. 119, C. V. Mosby, 1963.
4 EXAMPLE 3 Hydrolysis of polysaccharides The product obtained from Example 1 was hydrolized following the method of Wolfrom, Carbohyd. Res. (1969) 259-265. The product was dissolved in 72% sulfuric acid (w./w.) and left standing at 25 C. for two hours. It was then added to distilled water and refluxed for 2 /2 hours. The resulting acid mixture was neutralized with barium carbonate and passed over IR-l20 to remove the cation. The eluate was flash evaporated and the sample taken up in N-butanol:acetic acidzwater; 40:10:20 and separated overnight by paper chromatography. The presence of iduronic and glucuronic acid was established using sodium glucuronate and sodium idu' ronate as reference standards using dilute aqueous silver nitrate stain. Glucosamine was separated under similar hydrolytic conditions using one percent sulfuric acid. The mixture was neutralized with dilute aqueous sodium hydroxide and chromatographed on paper with the above described solvent system. Ninhydrin was used as the stain against authentic glucosamine as the standard.
FIGS. 1 and 2 are the IR and NMR curves, respectively, of the compound described in Example 1.
What is claimed is:
1. An anticoagulant which is a sodium salt of a polysaccharide, soluble in water, insoluble in acetone and ethanol, containing glucosamine, glucuronic acid and iduronic acid, having a molecular weight of 5300 daltons, containing by elemental analysis about 22% carbon, 3.6% hydrogen, 2.5% nitrogen, 11.4% sulfur, and 13.2% sodium the molar ratio C:N:S:Na being about 10:1:223; giving a positive metachromatic test, the free acid thereof and pharmacologically acceptable salts of said acid.
References Cited UNITED STATES PATENTS 2,959,598 11/1960 Doczi 260-209 R 3,262,854 7/1966 Yasuda 260211 R LEWIS GOTTS, Primary Examiner J. R. BROWN, Assistant Examiner US. Cl. X.R.