3,510,561 United States Patent Office Patented May 5, 1970 1. 2 3,510,561 of anticoagulant activity in the blood. Such repeated in SULFONE-ENHANCED HEPARN ABSORPTION jections are both inconvenient and painful. THROUGH MUCOUS MEMBRANES Teow Yan Koh, Toronto, Ontario, Canada, assignor to THE INVENTION Canada Packers Limited, Toronto, Ontario, Canada No Drawing. Continuation-in-part of applications Ser. No. 5 The invention in its broad aspect resides in the dis 457,501 and Ser. No. 457,502, both filed May 20, 1965, covery that certain sulfones enhance or promote the ab Ser. No. 550,935, May 18, 1966, and Ser. No. 686,323, Sorption of heparin through the mucous membranes, such Nov. 28, 1967. This application Aug. 18, 1969, Ser. No. as the Sublingual regions of the mouth and the walls of 851,102 the intestinal tract. By combining heparin with the sul Int, C. A61k 17/18 10 fone, the use of this highly regarded anticoagulant sub U.S. C. 424-183 26 Claims stance is extended to oral administration and to adminis tration to other mucous membranes. See U.S. Pat. No. 3,062,716 granted Nov. 6, 1962 for administration of ABSTRACT OF THE DISCLOSURE heparin rectally and U.S. Pat. No. 3,232,833, granted 15 Feb. 1, 1966, for administration of heparin to the mucous This invention relates to sulfone-enhanced heparin membranes of the eye, ear, nose and throat. absorption through mucous membranes and particularly The expression "oral administration' as used herein, to compositions and methods for administering heparin means administration by mouth and includes introduction so that it is absorbed through the mucous membranes to of the therapeutic compositions of the invention into the provide effective anticoagulant activity. The compositions 20 Sublingual or buccal regions for absorption therefrom, as contain heparin and at least one non-toxic physiologically well as the administration in the form of enteric-coated acceptable sulfone, such as a dialkylsulfone, a sulfolane or tablets or capsules for swallowing and subsequent release sulfolene. It has been discovered that these sulfones pro of the heparin in the intestine for absorption through the mote the absorption of the heparin anticoagulant sub intestinal walls. stance through the mucous membranes. The compositions 25 Use of the invention is in the fields for which heparin may also contain a fatty alcohol of chain length C14 to C24 therapy has already been established and may be in the to prolong or regulate the time period of absorption of veterinary field for the therapeutic treatment of animals. the active anticoagulant substance from the composition. The invention, in a further aspect, resides in the dis The composition may be administered in the form of covery that certain saturated and unsaturated fatty al solids or liquids which may be incorporated in enteric 30 cohols and mixtures thereof, preferably of chain length coated tablets or capsules for oral administration. C14 to C2a, when administered simultaneously with hep arin and a sulfone, improve and sustain the effectiveness of the anticoagulant activity. This represents a substantial The present application is a continuation-in-part of my advantage in anticoagulant therapy, where a sustained applications Ser. Nos. 457,501 and 457,502, filed May 20, 35 action is very desirable. Repeat dosages may be adminis 1965, now abandoned, my application Ser. No. 550,935 tered at longer time intervals and a more uniform re filed May 18, 1966 now abandoned and my application sponse attained. Ser. No. 686,323 filed Nov. 28, 1967. An object of the invention is to provide a heparin com 40 position which is effective when administered to the mu BACKGROUND AND DEFINITIONS cous membranes, including administration by the oral The invention relates to heparin compositions and route. methods of administration. Another object is to provide an improved heparin com The word “heparin' as used in the present application position from which the anticoagulant activity is absorb refers to the readily water soluble forms of the natural 45 able into the blood stream from the intestinal tract. sulfated polysaccharides composed of alternating hexos Another object of the invention is to provide a heparin amine and hexuronic residues including the non-toxic composition from which the anticoagulant activity is ab water soluble salts and water soluble derivatives thereof. sorbable from the intestinal tract over a Sustained period While the invention is exemplified with the sodium salt, of time. it is understood that other water soluble heparin com 50 Another object of the invention is to provide a heparin pounds known to the art, including other alkali metal salts composition which may be administered orally and from as well as organic salts, such as choline heparinate and which the anticoagulant activity can be slowly but con procaine heparinate may be used. See for example, U.S. tinuously absorbed through the mucous membranes. Pat. No. 3,062,716 granted Nov. 6, 1962, for a disclosure A still further object of the invention is the provision of of various heparin salts. 55 orally effective heparin compositions which are easy to Heparin has a long established reputation as a safe and encapsulate. effective anticoagulant. Its use, however, has been limited Suitable sulfones for the purposes of this invention by the need to administer it parenterally, since it is in are the non-toxic, pharmaceutically-acceptable sulfones active or only slightly active per se by routes other than which are substantially devoid of undesirable physiologi parenteral, for example by the oral route. (See, for ex 60 cal activity. Reference is made to U.S. Pat. No. 3,098,793, ample, the article by Windsor et al. entitled "An investi granted July 23, 1963, describing the known utility of gation of Routes of Administration of Heparin Other certain sulfones as pharmaceutical solvents for a wide Than Injection' in American Journal of Medicine, vol. variety of difficultly soluble medicinal agents. 37, September 1964, pages 408-416, wherein sublingual, Examples of the sulfones which enhance the absorp buccal, rectal and pulmonary routes using tablets, solu 65 tion of heparin through mucous membranes are those tions, suppositories and aerosols were reported.) Injec of the formula tions have been characterized by a quick response to pro RRSO vide a high level of systemic anticoagulant activity which wherein R1 and R2 are alkyl, alkylene, aralkyl, or to then declines at a rapid rate. Therefore, repeated intra gether form with the sulfur atom a saturated or unsatu venous injections or large subcutaneous injections are re 70 rated five or six membered ring such as in sulfolane and quired where it is desired to maintain a therapeutic level sulfolene. R1 and R2 may be the same or may be different. 3,510,561 3 4 Specific examples are the di-alkyl sulfones having the compositions of the invention is readily determined by formula: those skilled in the art. For example, sufficient of the composition is administered orally in one or more tab (I) R1 O lets or capsules to double the blood clotting time as deter N mined on a sample of withdrawn blood of the particular S species of mammal undergoing treatment. If the thera R^ O peutic level is to be maintained the dosage is repeated at intervals as deemed necessary. wherein R1 and R2 are alkyl or aralkyl (e.g. benzyl) Representative therapeutic dosages may contain, for groups having up to 12 carbon atoms. example, 50 to 100 mg. of heparin having an activity Examples of ring compounds are sulfoilane (tetrahydro O of 100 anticoagulant u/img., and from about 0.5-10grams thiophene 1,1-dioxide) having the formula: of the selected sulfone per 100 mg. of heparin. From 0.1 to 2 grams of fatty alcohol may be included. If water (II) O is included in the composition, the amount can vary 5 over wide limits, e.g. from 0 to 90%. The total daily unit of heparin for a mammal, such as a dog, may, for example, be from 50-1000 mg. (based (wherein a is a single bond), sulfolene (wherein a of on heparin having an activity of 100 u/mg.) in com Formula II. is a double bond), and their homologues and bination with from 0.5 to 10 grams of the sulfone and, close analogues, e.g. wherein one or more of the hy 20 if desired, from 0.1 to 10 grams of the fatty alcohol. drogen atoms may be substituted by hydrocarbon radi Because of the well-known instability of heparin in acids, cals as described in U.S. Pat. No. 2,360,861. standard enteric encapsulation procedure as set forth, for The fatty alcohols of chain length C14 to C4 are ex example, in Remington's Practice of Pharmacy, or in emplified by cetyl alcohol (C16), stearyl alcohol (C18), U.S. Pat. No. 3,126,320, granted Mar. 24, 1964, e.g. oleyl alcohol (C18), arachidyl alcohol (C20), behenyl 25 with cellulose acetate phthalate, or the like, is used to alcohol (C2) and mixtures thereof which are obtain provide an enteric capsule which when swallowed will able, for example, by reduction of corresponding fatty Survive the acid medium of the stomach and will be acids. The fatty acids themselves are ineffective. The dissolved in the alkaline medium of the intestine. As fatty alcohols of the chain length specified may either an example of this mode of administration, the dosage be saturated or unsaturated, e.g. oleyl alcohol. They 30 unit sufficient to double blood clotting time is given in possess good emulsifying properties. Further, it has been the form of one or more enteric-coated gelatin capsules. found that in general, the longer the chain length of the The invention is further illustrated by the following fatty alcohol, the better the degree of enhancement of examples of practice. the heparin composition with respect to sustained time of absorption of the anticoagulant activity. 35 EXAMPLE I The mechanism of the action of the sulfones is un 25 grams of diethyl sulfone (M.P. 73° C.) were dis known, but it is thought that they affect the porosity solved in 100 ml. of water to provide a 25 g. percent of the mucous membranes, e.g. the intestinal or sub solution which was adjusted by NaOH addition to a pH lingual mucosa, thereby permitting the absorption of 40 7.4. 100 mg. of heparin was then dissolved in 4 m. of heparin from the composition does not depend upon this solution. The resulting solution can be administered solubility characteristics either of the heparin in the by injection or to mucous membranes by known routes enhancing agent or of the enhancing agent in water. or may be encapsulated for oral administration. For the Heparin sodium, the commercial form of heparin, is latter, the amount of water can be decreased or omitted very soluble in water but cannot be absorbed from water entirely. solutions through the intestinal walls. On the other hand, 45 heparin sodium is not soluble in the sulfones but is ab EXAMPLE I sorbable from a composition containing a sulfone. (a) 25 grams of n-propylsulfone (M.P. 28-29° C.) The composition may be in the form of a solution or is mixed with 50 ml. of water and adjusted to pH 7.4 suspension or solid admixture of the ingredients depend 50 as in Example I. From 2 to 4 ml. of the resulting sus ing upon the solubility and melting characteristics of the pension is mixed with 100 mg. of heparin. selected sulfones. The presence of water is not critical (b) For encapsulation, the water in (a) may be and may be added if desired for convenience. It is pri omitted. Thus, 100 mg. of heparin are mixed with about marily used as a diluent, as a dispersing medium, and 2 grams of substantially neutral di-n-propyl sulfone to control dosages. In general, the amount of sulfone 55 Warmed to liquid form. The resulting suspension is present in the composition is the minimum that will pro cooled to Solid form, pressed into a tablet or encapsulated vide the desired adsorption-enhancing effect upon the in gelatin, following which the capsules are given a heparin. Where a C4 to C2 fatty alcohol is used in addi standard enteric coating, for example, with cellulose tion to the sulfone, the amount is usually at least equal acetate phthalate. on a weight basis to the heparin component. Function 60 ally, it may be expressed as that amount which will en hance and/or prolong the anticoagulant adsorption from EXAMPLE III the heparin-sulfone compositions. To 4 ml. of an aqueous n-propyl sulfone solution pre The composition may be compounds in a number of pared as in Example II, is added 200 mg. of a water ways. The two or three components as the case may 65 Soluble heparin derivative made by treating heparin be, may be simply mixed together with or without the Sodium with 0.15 NHCl in methanol to provide a partially addition of water. A convenient preparation, where the methylated and desulfated heparin assaying 70 U.S.P. fatty alcohol is used, is in the form of an emulsion. anticoagulant pl/mg. Thus, the fally alcohol may be thoroughly dispersed in the sulfone, the heparin or a water solution thereof O EXAMPLE IV added and the resulting material mixed until a uniform emulsion is formed. 100 mg. of heparin is mixed with 4 ml. of a solution In anticoagulant therapy, the desired therapeutic dos of 25 ml. of sulfolane in 25 ml. of water. This com age is the amount of heparin which is sufficient to dou position can be administered in the manner described in ble blood clotting time. The therapeutic dosage for the 75 Example I. 3,510,561 5 6 EXAMPLE V longed blood clotting time at the therapeutic level was, 100 mg.of heparin is dissolved in a solution prepared therefore, detected for 3 hours. by dissolving 25 grams of sulfolene (2,5-dihydrothio The effect of the sulfones and fatty alcohols upon the phene 1,1-dioxide) in 100 ml. of water. The product absorption of heparin through the intestinal wall is fur may be administered as previously described. ther illustrated by the following experiments on test ani mals. Two methods were employed as follows: EXAMPLE VI The compositions of Example I to V are prepared (A) Intestinal Loop. In Situ without the use of water, the proportions of sulfone Rabbits, of either sex, weighing approximately 3 kg. to heparin remaining approximately the same. These com 0 were used. Under ether anaesthesia, the abdomen was en positions are eminently suited for oral administration. tered through a mid-line incision. A suitable loop of the They are formed into powders or tablet for administra mid-gut, measuring about 6', was exposed and tran tion by the buccal route or encapsulated and/or enteric Sected, leaving the mesenteric blood supply intact. The coated for administration by swallowing and intestinal loop was then irrigated with aqueous sulfone solution or absorption. 15 suspension (ph 7.4) with which the heparin or heparinoid In a similar manner, heparin compositions are pro was to be instilled. The distal end was then ligated. The duced by the procedures of Examples I-VI using sub heparin in an aqueous solution or dispersion of the stituted compounds of the formula RRSO2 in lieu of selected sulfone was instilled into the loop and the diethyl and di-n-propyl sulfone, sulfolane and sulfolene. proximal end ligated. The closed loop was then replaced Mixtures of the sulfones can be employed beneficially 20 in the abdominal cavity. in lieu of a single sulfone. Thus, for example, sulfolane Blood samples (10 ml.) were taken by cardiac punc and n-propyl sulfone can be used together to enhance ture at intervals after instillation and the clotting time the absorption of heparin. determined by the method of Mayer, G. A., J. Lab. Clin. EXAMPLE VII 25 Med., 49, 938 (1957). 0.5 ml. of oleyl alcohol, 1.0 ml. of n-propyl sulfone (B) Intact in Vivo Intestine and 100 mg. of heparin are mixed together until an emulsion is formed. The heparin may be added in solu Ether-anaesthetized rabbits were used. The abdomen tion in e.g. 0.5 mi. of H2O if desired. was entered through a mid-line incision and the Ligament 30 of Treitz, identified. Aqueous heparin-sulfone solutions or In a similar manner, mixtures containing heparin or a dispersions, with added fatty alcohol in some instances, heparinoid, oleyl alcohol, cetyl alcohol, stearyl alcohol, were injected directly into the jejunum. behenyl alcohol or arachidyl alcohol and di-methyl Sul Clotting time of blood samples, taken at intervals fone, diethyl sulfone, di-n-butyl sulfone, sulfolane or after injection, was determined as previously described. sulfolene are produced. The test results are set forth below: EXAMPLE VII (1) Absorption of heparin in the rabbit 200 mg. of arachidyl alcohol are ground with 1.0 ml. of n-propyl sulfone until a homogeneous mass is pro intestinal loop in situ (sulfolane) duced. 200 mg. of a heparin derivative (prepared by 40 Sulfolane enhanced the absorption of heparin in the treatment of heparin sodium with 0.15 N HCl in meth intestinal loop in situ. The data which demonstrate the anol to provide a water soluble partially methylated and appearance of heparin in the blood stream as evidenced desulfated heparin derivative assaying 70 U.S.P. anti by the prolonged blood clotting time, are recorded in coagulant u/mg.) are added to the alcohol-sulfone mix Table . ture and the mass is stirred until a uniform emulsion is produced. 45 TABLE I-SYSTEMIC ANTICOAGULANT ACTIVITY In a similar manner, compositions are produced using Instillate Clotting time the other alcohols in lieu of arachidyl alcohol and/or the Hours.After Instillation other sulfones in lieu of n-propyl sulfone. The heparin Heparin, Sulfolane, - derivative may be added to the composition either as a ml. 6% 50 100 4 40'00' ---...-- solid or in the form of an aqueous solution, e.g. 200 mg. 13'00'’ of the heparin derivative readily dissolves in 0.5 ml. of 845 water, the resulting solution may be incorporated in the g/13 III alcohol-sulfone mixture. Solution.Heparin, 151 U.S.P. anticoagulant almg. Sulfonate, 50% aqueous EXAMPLE IX 55 Mean and S.D. of 20 rabbits. Effect of Oral Administration of Enteric-Coated Capsules of Heparin-Sulfone-Fatty Alcohol in Dogs The data presented in Table I show that a single dose A batch of enteric-coated capsules, each containing of a 4 ml. 50% aqueous sulfolane solution of 100 mg. 100 mg. sodium heparinate (159 U.S.P. anticoagulant 60 heparin to the intestinal loop resulted in at least 25 fold increase in clotting time. This effect took place 24 hours pl/mg.), 1.0 ml. di-n-propyl sulfone and 0.5 ml. oleyl after installation. The systemic anticoagulant activity alcohol was prepared. then fell and almost completely disappeared by 6% hours Three capsules were fed to a fasted, male dog weighing after instillation. 11 kg. Blood samples were taken at time intervals after Neither sulfolane nor heparin alone produced any anti feeding the capsules and the clotting time determined. 65 coagulation. Hours. After Feeding of Capsules (2) Absorption of heparin in the intact intestine Test O 2-3 3. 7 (sulfolane) Blood Clotting Time------520' 6027 1242/ 121? 612 70 Sulfolane enhanced the absorption of heparin in the in tact intestine. Therapeutically effective blood levels of heparin (twice the normal blood clotting time) were pro Following a lag period of 3 hours, the blood clotting duced when 6 ml. 50% aqueous sulfolane solution of time was elevated to 12°42'. By the 6th hour, twice the 100 mg. heparin was placed in the intact intestine. The normal blood clotting time was still maintained. Pro 75 data are presented in Table II. The effect was immediate. 3,510,561 8 Intravenous n-propyl sulfone produced no anticoagula TABLE ISYSTEMIC ANTICOAGULANT ACTIVITY tion indicating it had no anticoagulant activity. The con Material Injected Clotting Time Hours.After Injection trol experiment using n-propyl sulfone alone also did not Eeparin, Sulfoane, produce any anticoagulation. This would indicate that n mg. Iml. 0-k 2-6 propyl Sulfone did not cause the release of endogenous Rabbit No. 6. 100 6 84.543° 25'00' 1340' heparin into the blood. 00 6 8457-43. 28'00' 2112' It will be seen from Tables II and IV that 2-4 m. of 00 6 845-43. 2936' ------50 g. percent aqueous suspension of n-propyl sulfone with a ------6 845'43' 10'38' ------100 ------845'43' 8'50' 9'04 100 mg. of heparin provided a response in the intact in testine substantially equivalent to 6 ml. of 50% aqueous Erin, 151 U.S.P. anticoagulant p.fmg. Sulfonate, 50% aqueous O sulfolane with 100 mg. of heparin and that the response "'Sean and S.D. of 20 rabbits. after 3/2 hours was substantially as great for the n-propyl An intravenous injection of aqueous sulfolane did not Sulfone as that obtained after 2% hours with the sulfolane. produce any anticoagulation (data not included in Table Both materials are useful absorption enhancing agents. A II), i.e. sulfolane had no anticoagulant activity. The cont 5 single intra-jejunal dose of 2 ml. 50 g. percent suspension trol experiment using sulfolane alone (rabbit 9) also did of n-propyl sulfone and 100 mg. of heparin trebled the not produce systemic anticoagulant activity. This would blood clotting time after one hour. indicate that sulfolane did not cause release of endogenous A single intra-jejunal dose of 4 ml. 50 g. percent sus heparin into the blood. Heparin alone failed to be ab pension of n-propyl sulfone and 200 mg. of the heparin sorbed (rabbit 10). derivative almost doubled the blood clotting time in two hours and after three hours raised it approximately seven (3) Absorption of heparin from heparin-n-propyl sulfone fold. More prolonged activity is obtained by use of the in the rabbit intestinal loop in situ heparinoid as compared to heparin. n-Propyl sulfone enhanced the absorption of heparin Intraperitoneal injection of 2 ml. 50 g. percent aqueous in the isolated rabbit intestinal loop. The data presented 25 suspension of n-propyl sulfone produce no toxic symp in Table III demonstrate that a significant amount of toms in the rabbit, heparin was absorbed from the intestine into the blood, (5) Absorption of heparin in the rabbit intestinal loop raising its clotting time to above 180'. in situ (sulfolene) The control experiment, using n-propyl sulfone alone, An instillate of 100 mg. of heparin in 4 ml. 25 g. per produced no anticoagulation. Previous work had shown 30 cent Sulfolene to the ligated loop of the living anaesthe that heparin alone was not absorbed. tized rabbit raised the whole blood clotting time from a Gross examination at sacrifice of the animals showed normal value of 8 minutes 45 seconds to a value exceed no damage to the mucosa. ing 180 minutes, within an hour after introduction of the TABLE III-SYSTEMIC ANCOAGULANT ACTIVETY instillate. Significantly increased blood clotting time could AFTER INSTILLATION OF HEPARIN AND n-PROPYL 35 SULEONE INTO SOLATED JEJUNAL LOOP IN STU be maintained for approximately four hours. A control Instillate * experiment in which sulfolene alone was used did not o-m-m-m-m- Clotting Time Hours produce any anticoagulation. The results are shown in n-Propyl After Instillation the following table. Heparin, Sulfone, Rabbit No. Ing. ml. 0: 5 TABLE W.-SYSTEMIC ANTECOAGULANT ACTWITY 40 AFTER ADMINISTRATION OF HEPARN AND SU 00 4 845-43’ >90 ------FOLENE INTO SOLATED JEJUNAL LOOP N SITU 00 4 845'43’ >180 13357 3------4 8457-43, 905 852 Instiliate Clotting TimeHours after Instilation * Heparin, 151 U.S.P. anticoagulant plmg. n-Propyl Sulfone, 50 g. Rabbit Heparin, Sulfoiene, percent suspension. ml. 0: 1 22 4-6 ** Mean and S.D. of 20 rabbits. 4 845-43’ >90' 30 (4) Absorption of heparin from heparin-n-propyl 4 845'43’ >180 ------>60 4 845'-43’ >180' 45 ----- sulfone in the intact rabbit intestine 4 845'43' 90' ------848 n-Propyl sulfone enhanced the absorption of heparin Heparin, 155 U.S.P. anticoagulant p.fmg. Suifolene, 25 g. percent and of a heparin derivative in the intact rabbit intestine. 50 Solution. The data are presented in Table IV. Heparin accumulated * Mean and S.D. of 20 rabbits, in the blood to therapeutically effective level (twice the Sulfolene has no observable toxic qualities regardless normal blood clotting) within an hour after administra of the route of administration. tion. The systemic anticoagulant activity then fell, with (6) Systemic anticoagulant activity after administration the blood clotting time returning to the normal value 4 55 of heparin and diethyl sulfone to the isolated jejunal hours after administration. loop in situ TABLE IV.-SYSTEMIC ANTICOAGULANT ACTIVITY AFTER INTRA JEJUNUM ADMINISTRATION OF HEPARIN OR DERVATIVE AND nPROPYL SULFONE Injecta Cotting Time Hours After Injection Heparin, n-Propyl Rabbit No. ing. Sulfone, ml. to 2 3. 33 OO 4 845'43' 3250 6'05" 00 4 845/437 2835 35 00 3 845'43 29'53" 0207 O 2 845/43. 2649 920 00 2 845'43 220 929 w m a M - - - 4 845'43' 849 842 7------4 845'-43' 90' ------850 Heparin nPropyl derivative one, Ing. m 200 4 845-43' 445 1430' >60 ------200 4 845'--43' ------. 1530. 5320 --- 200 ------84543 800 745' ------* Heparin, 155 U.S.P. anticoagulant plmg. Heparin derivative, Partially methylated and desulfated heparinassaying at 70 U.S.P. anticoagulantu.fing. n-Propyl Sulfone, 50 g. percent suspension (pH 7.4). ** Mean and S.D. of 20 rabbits. 3,510,561 10 An instillate of 100 mg. of heparin in 4 mil. of 25 g. without stearyl alcohol, are presented in Table VIII. 100 percent diethyl sulfone to the ligated loop of the living mg, heparin and 2 ml. n-propyl sulfone in the absence of

TABLE VIII.-SYSTEMIC ANTICOAGUEANT ACTIVITY AFTER INTRA JEJUNUMSTEARYLALCOHOL ADMINISTRATION OF HEPARIN, n-PROPYL SULF ONE AND Injecta Clotting Time Hours. After Injection n-Propyl Stearyl Rabbit Heparin, Sulfone, Alcohol, No. mg. Inl. ng. 0 ki 3% 6 22 101------00 2.0 300 8’45-43' 54'13' ------25738 11754 102------100 2.0 300 845-43' 3545' ------48" 921 103------00 2.0 ------845-43' 32'50' 16'05'' ------104------00 2.0 ------8’45-43' 28'35' 13'15' ------Heparin, 155 U.S.P. anticoagulant pl.fmg. ** Mean and S.D. of 20 rabbits. anaesthetized rabbit raised the whole blood clotting time Stearyl alcohol resulted in heparin absorption so that the to a value exceeding 180 minutes within an hour after 20 clotting time was raised approximately four-fold, within introduction of the instillate. The clotting time was still 1 hour after administration. Following this, the systemic about treble normal clotting time after 5 hours. The re anticoagulant activity dropped abruptly with time. At 3% sults shown in the following table. hours after injection, the blood clotting time was reduced TABLE VI-SYSTEMIC ANTICOAGULANT ACTIVITY to approximately 14. The therapeutically effective level AFTER ADMINISTRATION OF HEPARIN AND DIETHYL 25 of blood heparin (twice the normal blood clotting time) SULEONE TO THE ISOLATED JEJUNAL LOOP IN SITU Was found to be maintained for approximately 2% hours. Ciotting Time Instillate Hours after Instillation When 300 mg. stearyl alcohol was added to the heparin Heparin, mg. n-propyl sulfone composition, better absorption of heparin Diethyl 30 occurred. The blood clotting time was raised approxi Rabbit No. - Sulfone, ml. 0 kt 5 mately six-fold. Furthermore, the maintenance of thera - 100 40 8/457-43’ >180 2440' peutically effective level of blood heparin was found to . . . . .00. . . . . 4.0 - 845-43’ >160 >30 4.0 845'--43' 8437 822 be of the order of approximately 16 hours. * Heparin, 155 U.S.P. anticoagulant pi.fmg. Diethyl Sulfone, 25 g. (9) Heparin-n-propyl sulfone-arachidy alcohol percent solution, pH 7.4. - 35 ** Mean and S.D. of 20 rabbits. The appearance of systemic anticoagulant activities of 3 (7) Systemic anticoagulant activity after administration Irabbits, as evidenced by the increased blood clotting time, of heparin and diethyl sulfone to the intact intestine after intra-jejunum administration of heparin and n-pro In the intact intestine the blood clotting time was sig- 40 cordedpyl sulfone in Table with IX. and without arachidyl alcohol are re

TABLE X SYSTEMIS ANTICOAGULANT ACTIVITY AFTER INTRA ARACEIDYLMADMINISTRATION ALCOHOL OF HEPARIN,n-PROPYLSULFONi AND Injecta Clotting Time Hours After Injection n-Propyl Arachidyl Rabbit Heparin', Sulfone, Alcohol, - No. Ilg. ml. Ing. 0k 3% 4% 6 "H------uuluul

105------100 1.0 200 845'43' 63'32" . 2823 1945 106.----- 100 10 ------8’45'43' 26'49' 920 psa ------4s 107------100 1.0 ------8’45'--43' 22/10" 929 IIII Reparin, 155 U.S.P anticoagulant u?mg. **Mean and S.D. of 20 rabbits. nificantly increased by the administration of 100 mg. of That data in Table IX indicate that 100 mg. heparin heparin in 8 ml. of a 25 g. percent solution of diethyl and 1.0 ml. n-propyl sulfone administered intra-jejunally sulfone. The results are shown in the following table. to the rabbit raised the blood clotting time approximately TABLE VII-SYSTEMIC ANTICOAGULANT ACTIVITY 60 AFTER ADMINISTRATION OF HEPARIN AND DIETHYL three-fold, within 1 hour after administration. It was SULF ONE TO THE INTACT INTESTINE found that the maintenance of therapeutically effective Clotting Time level of blood heparin was of the duration of approxi Injecta Hours after Injection mately 2 hours. Diethyl Rabbit Heparin, Sulfone, 65 With the addition of 200 mg. arachidyl alcohol, the ab No. 3. ml. 0 #8 34 22 5 Sorption of heparin was improved. The blood clotting l------100 8.0 - 845-4-43' 843' 1430' 710 time was raised to 7 times above normal. The maintenance 2------8.0 c. 845'--43 -10'05'. 8'47' ------... of therapeutically effective level of blood heparin was also - * Heparin, 155. US.P. anticoagulant pri?mg. Diethyl Sulfone, 25g. prolonged. It was maintained for a period of at least 6 percent solution, p 7.4. . . . ** Mean and S.D. of 20 rabbits. 70 hours. (8) Heparin-n-propyl sulfone-stearyl alchol (10) Heparin-n-propyl sulfone-behenyl alcohol - The systemic anticoagulant activities of 4 rabbits, as ... The rabbits' responses, in terms of blood clotting time, measured by the blood clotting time, after intra-jejunum after intra-jejunum injection of heparin, n-propyl sul administration of heparin and n-propyl sulfone with and fone and behenyl alcohol are recorded in Table X. 3,510,561 11 12 TABLE X-SYSTEMIC ANTICOAGULANT ACTIVITY AFTER INTRA-JEJUNUM. ADMINISTRATION OF HEPARIN, BEHENYL ALCOHOL AND n-PROPYL SULFONE Injecta, Behenyl n-Propyl Cotting Tine, Hours after Injection Rabbit Heparin, lcohol, ulfone, 0. Ing. Ing. ml. 440 3 4. 6 100 200 2.0 845'--43' >315 - 810 - 100 200 2.0 845'43' 1162 -- 1351 - 100 200 1.0 845'43' 1630'------'35 100 200 1.0 845'43' ------5430' . 00 200 1.0 845'43'- - 1240' ------00 200 1.0 84.543 a ma w w u a w w w ------...... 10'47' ------Approximate. " Heparin, 155 U.S.P. anticoagulant pi.fmg. ** Mean and S.D. of 20 rabbits. If the data (rabbits 110-113) of Table X are compared after intra-jejunum administration of heparin, n-propyl to the data for the heparin-sulfone alone (rabbits 106, 5 Sulfone and oleyl alcohol, are recorded in Table XI. 107) of Table IX, it is noted that behenyl alcohol greatly Oleyl alcohol, in common with the saturated alcohols enhanced the adjuvant action of n-propyl sulfone. The (C18, Cao and Ca2) also enhanced the adjuvant action of peak anticoagulant activity was raised to 54' 30'. The n-propyl sulfone. Compared to the data for n-propyl sul same quantity of n-propyl sulfone promoted heparin ab fone-heparin alone, it is observed that oley alcohol add sorption with the blood clotting time raised to an average 20 ed to heparin and n-propyl sulfone increased the blood of only 24' approximately. Two hundred mg. behenyl al clotting time to approximately 130'. The maintenance of cohol also sustained therapeutically effective level of therapeutically effective level of blood heparin was also blood heparin for approximately 5 hours. in contrast to 2 further improved to approximately 4% hours. TABLE XI.-SYSTEMIC ANTICOAGULANT ACTIVITY AFTER INTRA JEJUNUMADMINISTRATION OF HEPARIN, n-PROPYL SULF ONE AND OLEYL. ALCOOL Injecta Clotting Time Hours.After Injection Oley n-Propyl Rabbit Heparin", Alcohol, Sulfone, No. Ing. ml. ml. 0.4 1. 5 19 114------100 0.5 1.0 845-43 Q140 ------8387 l6------100 0.5 1.0 845'43' 127457 'i30' ------Approximate. Heparin, 155 U.S.P. anticoagulant. *Mean and S.D. of 20 rabbits. hours when n-propyl sulfone alone was used. The pro (12) Sulfolane heparin-behenyl alcohol longed maintenance was associated but not coupled to increased anticoagulant activity. This is evident from the The responses on administration of heparin, sulfolane data in Table X. If the volume of n-propl sulfone was in and behenyl alcohol are shown in the following table: TABLE XII.-SYSTEMIC ANTICOAGULANT ACTIVITY AFTER ADMINISTRATION OF HEPARIN, SULFOLANE AND BEHENYL ALCOHOL TO THE INTACT INTESTINE Injecta * Clotting Time Hours after Injection Behenyl

Heparin, Sulfolane, Alcohol, g. Inl. Ing. to 2 2-3 4. 6 100 3.0 - 8’45'437 25'00'’ - 1340 00 3.0 - 8’457-43. 28'00'’ 2127

100 3.0 - 8’45'437 2936 - - - 10 3.0 500 845'43' ------0.357 *Heparin, 151 U.S.P. anticoagulant filmg. **Mean and S.D. of 20 rabbits. creased to 2.0 ml. and the amounts of heparin and be From the data in Table XII it will be seen that heparin henyl alcohol kept constant (rabbits 108, 109), in the in and sulfolane alone provided a therapeutic level of hep pecta, the blood clotting time was raised to about 315 55 arin after one hour and that this was maintained for about and 162 respectively, yet the maintenance of therapeuti 1-2 hours. Addition of behenyl alcohol greatly enhanced cally effective level of blood heparin was maintained for the blood clotting time, giving an approximate eleven-fold only 3 hours. A comparison of the data (rabbits 108, 109 increase after two hours and therapeutic levels were compared to rabbits 110-113) would indicate that pro maintained for at least three hours. longed maintenance was not necessarily a consequence of increased anticoagulant activity. The emulsions admin 60 (13) n-Propyl sulfone-heparin derivative-arachidyl istered to rabbits 110-113 were more viscous than the alcohol emulsions administered to rabbits 108 and 109. The response on administration of partially methylated (11) HeparinX-n-propyl sulfone-oleyl alcohol and desulfated heparin, with n-propyl sulfone and arachi The rabbits' responses, in terms of blood clotting time, dyl alcohol is shown in the following table. TABLE XII.-SYSTEMIC ANTICOAGULANT ACTIVITY AFTERADMINIS TRATION OF A HEPARIN DERIVATIVE, n-PROPYL SULF ONE AND ARACEDY ALCOHOL TO HE INTACT INTESTINE Injecta" Clotting Time Hours after Injection Heparin n-Propyl Arachidyl derivative, Sulfone, Alcohol, Rabbit No. Ing. e Ing. st 0 34 6 232 120------200 1.0 200 874543' 1220 225' 330 "Heparin Derivative, Partially methylated and desulfated heparin assaying at 70 U.S.P. anticoagulant fing. **Mean and S.D. of 20 rabbits, 3,510,561 13 14 It will be seen from Table XIII that the blood clotting amount effective to regulate the time period of absorption time was substantially increased after 3/2 hours and Was of the anticoagulant substance from the composition. still increasing after 23/2 hours. 5. A therapeutic composition in enteric-coated oral In each of the foregoing experiments 8-13, the heparin pharmaceutical capsule form containing an effective was dissolved in 0.5 ml. of water which served as a diluent anticoagulant dosage of a water soluble form of heparin to provide a more fluid composition and facilitate injec in combination with an amount of a non-toxic physio tion into the tract of the experimental animals. The hep logically-acceptable, sulfone selected from the group arin solution was incorporated with the selected sulfone consisiting of diethyl sulfone, di-n-propyl sulfone, sul -in the manner set forth in Examples VII and VIII. folene, sulfolane and mixtures thereof, effective to en (14) Intestinal absorption of encapsulated heparin-sul 0 hance the absorption of heparin anticoagulant substance fone-oral administration versus intra-jejunum injection through the mucous membranes. 6. The composition of claim 5 wherein there is in in the dog cluded a fatty alcohol selected from the group consisting Two enteric-coated gelatin capsules, each containing of cetyl, alcohol, arachidyl alcohol, oleyl alcohol, stearyl 100 mg. sodium heparin in 1.0 ml. of di-n-propyl sulfone alcohol, behenyl alcohol and mixtures thereof in an were fed simultaneously to a female dog (weight 11 kg.), amount effective to regulate the time period of absorp which had been fasted overnight. Blood samples were tion of the heparin anticoagulant substance from the taken by venepuncture at intervals after feeding and the composition. respective clotting times were determined. 7. The composition of claim 5 wherein the anti After a 4-day recovery period, an intra-jejunum injec 20 coagulant is heparin and the sulfone is diethyl sulfone. tion of an equal amount of sodium heparin and di-n- 8. The composition of claim 5 wherein the anti propyl sulfone as was contained in the two capsules was coagulant is heparin and the sulfone is di-n-propyl administered to the same dog and blood samples were sulfone. taken at intervals and clotting times determined. The re 9. The composition of claim 5 wherein the anti sults are given in the following Table XIV. 25 coagulant is heparin and the sulfone is sulfolane. TABLE XIV.-SYSTEMIC ANTICOAGULANT ACTIVITY OF 10. The composition of claim 5 wherein the anti HEPARIN-SULFONE IN DOG-ORALLY WS, INTRA coagulant is heparin and the sulfone is sulfolene. JEJUNAL INJECTION 11. The composition of claim 6 wherein the sulfone Clotting Times Hours after Administration is di-n-propyl sulfone and the alcohol is oleyl alcohol. 30 12. A method for the administration of heparin to 0 1. 2 23 3 3% 4% 5 mammalian animals comprising orally administering at Oral------5% ------10 ------11 7 6 least one enteric-coated capsule containing an effective Intrajejun --- 5%. 11%. 10 ------5% ------anticoagulant dosage of a water soluble form of heparin in combination with an amount of a non-toxic, physi Systemic anticoagulant activity, at approximately ther 35 ologically-acceptable sulfone selected from the group apeutic level appeared after about 2% hours on oral ad consisting of diethyl sulfone, di-n-propyl sulfone, sul ministration in capsule form as contrasted to 1 hour upon folane and Sulfolene sufficient to cause effective absorp intra-jejunal injection. In each instance approximately tion of anticoagulant activity through the mucous mem this level of activity was maintained for about one hour branes into the blood stream. before dropping off to normal. This experiment indicates 40 13. The method of claim 12 wherein the orally effec that approximately the same level of therapeutic activity tive anticoagulant dosage also contains a fatty alcohol is to be expected whether the composition is introduced Selected from the group consisting of cetyl alcohol, ara into the intestine orally in the form of enteric-coated cap chidyl alcohol, oleyl alcohol, stearyl alcohol, behenyl sules or directly into the jejunum by injection, substan alcohol and mixtures thereof in an amount effective to tially the only dieffrence being in the time lag required regulate the time period of absorption of the anticoagul for the capsules to reach the intestine. 45 lant Substance from the composition. It is to be understood that various changes may be 14. The method of claim 13 wherein the sulfone is made in the proportions of the ingredients in the com di-n-propyl sulfone and the fatty alcohol is oley1 position without departing from the spirit and scope of alcohol. the invention. 50 15. A therapeutic composition comprising an effective I claim: anticoagulant dosage amount of heparin sodium in com 1. A therapeutic composition from which heparin anti bination with at least one non-toxic, physiologically ac coagulant substance is absorbable through mucous mem ceptable Sulfone having the formula RRSO, where R branes comprising an effective anticoagulant dosage and R2 are alkyl, alkenyl or aralkyl of up to 12 carbon amount of a readily water soluble form of heparin in 55 atoms or together with the sulphur atom form a ring combination with at least one non-toxic, physiologically having four ring carbon atoms, said sulfone being pres acceptable sulfone having the formula RRSO where R1 ent in an amount effective to enhance the absorption and R2 are alkyl, alkenyl or aralkyl of up to 12 carbon of the heparin anticoagulant substance through the mu atoms, or together with the sulfur atom form a ring cous membranes. having four ring carbon atoms, said sulfone being present 60 16. A therapeutic composition in enteric coated oral in an amount effective to enhance the absorption of the pharmaceutical capsule form containing an effective heparin anticoagulant substance through the mucous anticoagulant dosage of heparin sodium in combination membranes. With an amount of a nontoxic, physiologically acceptable 2. The composition of claim 1 wherein there is in Sulfone selected from the group consisting of diethyl cluded a fatty alcohol of chain length C14 to C2 in an 65 Sulfone, di-n-propyl sulfone, sulfolene, sulfolane and the amount effective to regulate the time period of absorp mixtures thereof, effective to enhance the absorption of tion of the heparin anticoagulant substance from the the heparin anticoagulant substance through the mucous composition. membranes. 3. The composition of claim 1 wherein the sulfone is 17. The composition of claim 16 wherein there is selected from the group consisting of diethyl sulfone, di 70 included a fatty alcohol of chain length C to C24 in n-propyl sulfone, sulfolene, and mixtures thereof. an amount effective to regulate the time period of absorp 4. The composition of claim 3 wherein there is in tion of the heparin anticoagulant substance from the cloded a fatty alcohol selected from the group consisting composition. of cetyl alcohol, arachidyl alcohol, oleyl alcohol, stearyl 18. The composition of claim 16 wherein there is alcohol, behenyl alcohol, and mixtures thereof in an 75 included a fatty alcohol selected from the group con 3,510,561 15 6 . . . sisting of cetyl alcohol, arachidyl alcohol, oley alcohol, selected from the group consisting of cetyl-alcohol, ara steary alcohol, behenyl alcohol and mixtures thereof in chidyl alcohol, oleyl alcohol, stearyl alcohol, behenyl an amount effective to regulate the time period of absorp alcohol and mixtures thereof in an amount effective to tion of the heparin anticoagulant substance from the regulate the time period of absorption of the anticoagul composition. lant substance from the composition. 19. The composition of claim 16 wherein the sulfone 26. The method of claim 25 wherein the sulfone is is diethyl sulfone. di-n-propyl sulfone and the fatty alcohol is oleyl alcohol. 20. The composition of claim 16 wherein the sulfone is di-n-propyl sulfone. References Cited 21. The composition of claim 16 wherein the sulfone O UNITED STATES PATENTS is sulfolane. 2,656,298 10/1953 Loewe ------424-183XR 22. The composition of claim 16 wherein the Sulfone 2,805,977 9/1957 Robinson et al. ------424-19 is sulfolene. 2,875,130 2/1959 Grass et al. ------424-19 23. The composition of claim 16 wherein the sulfone 3,088,868 5/1963 Windsor ------424-183 XR. is di-n-propyl sulfone and the alcohol is oleyl alcohol. 5 24. A method for the administration of heparin to 3,098,793 - 7/1963 Loew ------424-115 mammalian animals comprising orally administering at 3,126,320 3/1964 Morii et al. ------424-183XR least one enteric coated capsule containing an effective 3,146,167 8/1964 Lantz et al. ------424 -19 anticoagulant dosage of heparin sodium in combina 3,181,996 5/1965 Bianchini------424-183 tion with an amount of a nontoxic, physiologically 20 3,232,833 2/1966 Riviere ------424-183 acceptable sulfone selected from the group consisting of OTHER REFERENCES diethyl sulfone, di-n-propyl sulfone, di-n-propyl sulfone, sulfoane and sulfolene sufficient to cause effective ab Windsor et al., Amer. J. Medicine 37:408-416, Septem sorption of anticoagulant activity from the heparin sodi ber 1964, "In Investigation of Routes of Administration um through the mucous membranes into the blood of Heparin Other Than Injection.” Y stream. S. K. ROSE, Primary Examiner 25. The method of claim 24 wherein the orally effec tive anticoagulant dosage also contains a fatty alcohol