METHOD OF INHIBITING COAGULATION OF BLOOD

Abstract: A method for inhibiting or preventing coagulation of blood by inhibiting platelet aggregation. More specifically, a method is disclosed for inhibition of a platelet aggregation by the administration of a compound having as part of its molecule the structure [1,4 dioxo-2-ene] also designated as [oxo-ene-oxo]. Among disclosed exemplary inhibitory compounds are 1,4 dioxo-trans-2-ene-derivatives of long chain fatty acids, but long chain structure is not essential.

Description: METHOD OF INHIBITING COAGULATION OF BLOOD The present invention is directed to a method for the inhibition or prevention of blood coagulation by the inhibition of blood platelet aggregation. More specifically, the present invention is directed to the inhibition of blood platelet aggregation by a compound having as part of its molecule the structure [l,4-dioxo-2-ene] , also designated herein as [oxo- ene-oxo]. Of such compounds, 1 ,4-dioxo-trans-2-ene- derivatives of long chain fatty acids are exemplary, although the long chain structure is not essential to the invention. Generally, the clotting of blood has the benefi¬ cial effect of minimizing loss of blood from ruptured blood vessels whether the result of an open wound or an internal disruption of the vessels. However, when clotting hinders blood flow through various internal organs and tissues, it may have detrimental and life-threatening effects, such as thrombosis, myocar- dial infraction, stroke, or renal damage. Aggregation of blood platelets, either by interaction with collagen exposed by rupture of blood vessel subendothelial layers or by interaction with thrombin, has been recognized as an early and very important phase in the cascade of events which ultimately results in blood clot formation. As soon as a small number of platelets become aggregated, they begin the release internally stored aggregating factors, among them serotonin and 5'-adenosine diphosphate, which cause an ever increasing number of additional platelets to aggregate. This with the involvement of erythrocytes and fibrin, eventually leads to the formation of a hemostatic plug in the blood vessel. In view of its autocatalytically promoting effect, platelet aggregation has been extensively studied with the goal of controlling or preventing the formation of a blood clot at this early point of development. Platelet aggregation may be induced physiolog¬ ically by the interaction of circulating platelets in the blood with either collagen or thrombin, the latter arising from prothrombin by enzymic hydrol¬ ysis. Interaction of platelets with collagen or thrombin culminates in the formation of platelet generated products which arise from enzymic oxygena- tion of free arachidonic acid. These oxygenated products, i.e., PGG^, -PGH ? and TXA~ , are potent platelet aggregating agents. Various agents are known to inhibit both the extrinsic and intrinsic blood clotting pathways by affecting inhibition of the proteolytic conversion of prothrombin to thrombin. Other platelet aggregation- preventing agents intervene in the liberation of free arachidonic acid released from the lipids of plate¬ lets or other bodily tissues; among these are ster- oidal anti-inflammatory agents (glucocorticoids) , compounds of the phenolthiazine-type (m-chloroproma- zine) , and other cationic anti-malarial drugs (chloroquine) , which impair calcium functions. Other inhibiting agents irreversibly block the oxidizing enzyme which converts free arachidonic acid into the aggregation-active prostaglandin endoperoxides. However, products from action of this oxygenase can be transformed also into compounds which counteract aggregation or have other important physiological functions. Aspirin and indomethacin are examples for such ambivalent effects. They block the action of cyclooxygenase on arachidonic acid and thereby the formation of the aggregatory, TXA-, but they also block production of the anti-aggregatory, PGI 2 . In addition to this particular dilemma concern¬ ing arachidonic acid, the oxygenases also act upon other polyunsaturated fatty acids which are similar to and in nature closely associated with arachiodonic acid. Products from them widen the array of physi¬ ologically active compounds which must be in delicate balance for the healthy state of the tissue and organism. The complexity is further deepened—when considering that each kind of tissue produces from arachidonic and related acids its own profile of active substances which can be classified as local hormones. Inhibitory drugs targeted specifically at one tissue, one of its oxygenases, one polyun¬ saturated fatty acid and one or selected products from it have not yet been found. The present invention is directed to a novel and useful approach to the regulation of blood clotting by the use of a specific family of compounds, which block platelet aggregation, and which do not prevent the oxidative conversions of arachidonic acid in the clotting cascade. Broadly stated, the present invention comprises a method of inhibiting or preventing coagulation of blood by effectively inhibiting the aggregation of blood platelets by compounds which contain a system of oxo and trans-double bond functions as a charac¬ teristic feature of their molecular structure. The compounds according to the present invention which have the highest inhibitory activity are these which have the conjugated system;-[1,4-dioxo-trans-2-ene]- as part of their structure; this family may be represented by the structural formula: wherein R. and 2 are C-3 to C-10 aliphatic substi- tuents, which may be straight or branched chain aliphatics, which may be the same or different, and which may optionally have additional chemical func¬ tions, such as carboxyl, carboxyl ester, alkoxyl or the like, with or without double bonds. The compounds according to the present invention which have been found to have the highest activity in preventing aggregation of blood platelets are those compounds having the conjugated system:-[1,4-dioxo- trans-2-eno]- (-]oxo-ene-oxo]-) as a characteristic feature of their molecular structure. Such compounds are represented by the structural formula: H R 1 -[C-C=C-C]-R 0 l n ι 'i 2 O H 0 wherein R- and R_ are C-3 to C-10 aliphatic substi- tuents, straight or branched, which may be the same or . different, and which may optionally have additional chemical functions. Specific examples of R- and R 2 substituents according to the present invention are shown in the following table: TABLE I Compound R_. R« -C 7-H1.4.COOH -C_6.H n 13. -C 7 H 14 COOCH 3 " C 6 H 13 -C 8 0 H1 η 6,-COOH -C5-H1-1. -C 8 H 16 COOCH 3 -C 5 H -C 9 H 18 COOH -C 6 H 13 -C 6 H 14 COOCH 3 -CH=CHC 4 H 9 7* -C 8 (OCH 3 )-C 5 H 10 COOCH 3 _ C6 H 13 ■ C-,H 14 (COOCH 3 ) "C 3 H 7 -C 3 H ? r ftιixture of positional isomers The inhibitory compounds, according to the present invention and carrying the substitutions shown in Table I, have individual biological activi¬ ty, as measured by IC j . Q , of between lχ.M and lOJl. These compounds, examples of the compounds of the present invention, include 9,12-dioxo-trans-10- octadecenoic acid (compound 1) and its methyl ester (compound 2); 10; 13-dioxo-trans-ll-octadecenoic acid (compound 3) and its methyl ester (compound 4) ; ll,14-dioxo-trans-12-eicosenoic acid (compound 5 - the bis homologue of compound 1) ; 4,7-dioxo-trans-5- decene (compound 8) ; and the olefinic (compound 6) an methoxyl substituted (compound 7) compounds. The biological activity measured by IC,- n of the latter two compounds is about 2^J_. Hydrogenation of the double bond shown in Formula I renders these compounds inactive. As indicated by the exemplary compounds shown in Table I, besides the specified core structure which gives these compounds the desired activity, the compounds may carry additional functional - groups as, for example, a primary carboxyl or ester groups. We have also found that the relative position of the con¬ jugated system in the overall molecule can be changed without loss of inhibitory activity. Besides the oxo-ene-oxo structure conferring activity, these examples are characterized by R. and R- groups of low polarity. They provide for solubil¬ ity in solvents of low or moderate polarity and therefore correlate with accessibility to biological membranes. Compounds 1 to 7, for example, can be expected to resemble fatty acids and esters in their metabolism and catabolism; Compound 8 has hydro¬ carbons as R 1 and R 2 , therefore its metabolism and catabolism can be expected to be slower and utilized by a different biological pathway. This difference provides the choice of synthesizing specific inhibi¬ tors according to the present invention having one or the other chemically rather simple substitutions in contrast to the vast majority of previously known inhibitors which are complex, unpredictable in regard to metabolism and catabolism, and which may result in undesirable metabolic products. In order for the person skilled in the art to which this invention pertains to achieve a better and more thorough understanding of the invention, the following examples are provided. EXAMPLE I The effect of dioxo-ene compounds on thrombin induced platelet aggregation was studied. Incuba¬ tions of 0.5 ml volumes at 37° contained 20 M JA test acids in 15 mM tris buffer at pH 7.5, 140 mM NaCl, g 5.5 mM glucose and 1 to 3 x 10 platelets. CAC1 2 , 2 mM final concentration, and i min later 0.2 units thrombin from human plasma, were added to induce aggregation, and the change of light transmission was recorded, the increase of light transmission being a measurement of aggregation. The results of this inhibition are shown in Figure 1. The control experiment, Panel I of Figure I, established that the platelets were functional under commonly accepted conditions; this was further ascertained by the fact indomethacin exerted its well known aggregation inhibitory effect (shown in Fig. 1) . Panel II of Figure 1 shows that 9,12-dioxo-trans-10-octadecenoic acid inhibits aggregation whereas the stereoisomeric cis compound does not. Panel III shows inhibition by 10,13-dioxo-trans-ll-octadecenoic acid, a positional iso er of the inhibitory compound in Panel II. When a methyl (Me) group is a substituent at the double bond the compound is not inhibitory. 9,12-dioxooctadecenoic acid, a compound carrying two oxo groups in appropriate positions of the molecule but without the double bond, was found to be inactive. In further variation, but retaining the active portion of the structure, 11,14-dioxo- trans-12-eicosenoic acid and the methyl ester of 9 ,12-dioxo-trans-ll-octadecenoic acid were found to be inhibitory. This confirms the surmise that other features of the molecular structure can be varied without losing the inhibitory activity. EXAMPLE II The response of platelet aggregation induced by thrombin to varying dosages of the inhibitors was studied, and the results are shown in Figure 2. Except for the concentrations of the inhibitory acids, experimental conditions were as described in Example I. 9,12-dioxo-trans-10-octadecenoic (upper panel) and 10,13-dioxo-trans-ll-octadecenoic (lower panel) acids were evaluated. Such dosage experiments determine the concentration necessary for 50% inhibi¬ tion (IC ςf) ) , a parameter commonly used for expressing the inhibitory effectiveness of a compound.. This value for the newly found inhibitors is between 1 and 6^i. The literature quotes IC- 0 values for inhibitors of oxygenases in a range from 0.26 y (mefenamate) to 176 yϋ (aspirin) and higher concentrations. These values refer to oxygen uptake of σyclooxygenase or lipoxygenase or of both, or refer to amounts of specific products from arachidonic acid which cause platelet aggregation, but they do not describe the effectiveness to inhibit aggregation in the presence of such agents. Since the mode of action of the newly found inhibitors of platelet aggregation is distinct from that of cyclooxygenase inhibitors, no quantitative comparison of strength of the new inhibitors can be made with the inhibitors of the oxygenases or of other reactions. One can state, however, that IC_ n for aggregation between 1 and 6 Λ M, as in Example II, is not only at the low range of the concentrations quoted for other inhibitors but also characterizes the efficiency of the compounds in closer relation than those of the literature to the medically desired activity against thrombin and arachidonic acid induced aggregation. EXAMPLE III The response of platelet aggregation induced by arachidonic acid to varying dosages of the same inhibitors as in Example I was studied and the results are shown in Figure 3. Experimental condi¬ tions were as described in Example I except that concentrations of the inhibitory acids were varied and that platelet aggregation was induced by 20 t-M potassium arachidonate. The inhibitors were 9,12- dioxo-trans-10-octadecenoic (upper panel) and 10,13- dioxo-trans-11-octadecenoic (lower panel) acids. The IC gQ value also is between 1 and 6 _ / . EXAMPLE IV The effect of increasing preincubation periods on inhibition of aggregation induced by thrombin was studied and the results are shown in Figure 5. The conditions of aggregation were as described in Example I. Platelets were preincubated for varying short periods of time with 8 #/* < 4,7-dioxo-trans-5- decene before addition of thrombin. Varying the preincubation period revealed that the inhibitory potency is increased with increasing periods of preincubation. Preincubation in vitro reflects conditions to be encountered in the body. EXAMPLE V Inhibition of aggregation caused by thrombin, as well as by arachidonic acid, suggests that these inhibitors act at a late step in the course of events and that the normal enzyme reactions of arachidonic acid are not affected. Indeed, experimentation determined that the oxygen consumption of arachidonic acid catalyzed by cyclooxygenase or lipoxgenase was not changed by inhibitors in 15 to 120^—M concentra¬ tions. In addition, investigation of the products from [1- 14C]arachiodonic acid with platelets demon¬ strated that the inhibitors do not affect the course of the reactions which normally follow the oxygena- tions catalyzed by the platelet enzymes. Incubations were for 5 minutes under conditions described for Example V, in 3 ml volume. The substrate acid, [1- 14C]arachidonic acid (200,000 cpm) and the test acids were at final concentration of 20^ Λ. Analyses of products were by 14C-TLC scanning, with appropriate reference for identification. The results are given in Table II. Data are averages of duplicate experiments. TABLE II Products from arachidonic acid incubated with plate¬ lets in presence of non-inhibiting 9,12-dioxoctade- canoic and of aggregation inhibiting 9,12-dioxo-trans-10-octadecanoic acids. P R 20^M 20:4 + 20 ^ ^ 20:4 + 0 D 20 cM 20:4 20 s M 9,12- 20 *M 9,12- U C (control) dioxo-18:0 dioxo-trans- T 11-18:1 s cpm - TXB. 39,980± 540 38,170± 170 35,170± 10 HHT 45,680± 290 42,940± 1,250 39,450+ 1,500 HETE 82,210± 5,090 69,730+ 940 74,700+ 500 20:4 21,250± 4,370 13,420± 3,340 10,670+ 1,150 Table II lists the major products from cycloox- genase activity, i.e., 12-hydroxyheptadecatrienoic (HHT) acid and the prostanoid T B 2 , and from lipoxy- genase activity, 12-hydroxyeicosatetraenoic (HETE) acid. Their amounts are not changed by presence of antiaggregatory -[1,4-dioxo-trans-2-ene]- compounds. As expected, the above-mentioned inactive dioxo compound without double bond does not affect the oxygenation reactions either. Ambivalent effects with aspirin and indomethacin are not likely to be caused by the dioxoene inhibitors. Formation of the prostanoid TXB 2 from arachi¬ donic acid by platelet cycloxygenase (Table I) involves the short-lived intermediates PGG-, PGH 2 and TXA 2 which are known to strongly promote platelet aggregation, although they are very rapidly further metabolized. Apparently, none of them can exert much activity in the presence of the inhibitors. EXAMPLE VI The effect of 9,12-dioxo-trans-10-octadecenoic acid on the thrombin stimulated formation of phospha- tidic acids was studied. Washed ^-labeled plate- g lets (0.35 x 10 cpm) were incubated for 5 minutes with thrombin under conditions as described for Example V, but enlarged to 4 ml volume. Analyses of products were by "-TLC scanning, with appropriate references for identification. Data in Table III are averages of duplicate experiments. TABLE III Formation of Phosphatidic Acid PI PA Treatment (cpm x 10 -3) (cpm x 10-3) 32 P-Platelets 588 ± 20 55.6 ± 5 (reference) + * Thrombin 641 ± 5 537 ± 25 + Thrombin + 9,12-dioxo-18:0 774 ± 78 533 + 16 + Thrombin + 9,12-dioxo-trans- 10-18:1 761 ± 12 486 + 32 Table III shows the lack of inhibitory effect of 9,12-dioxo-trans-10-octadecenoic acid on the thrombin stimulated formation of phosphatidic acids from prelabeled phospholipids of intact human platelets. These findings suggest that the described inhibitory compounds of platelet aggregation do not intervene at the platelet receptor level. EXAMPLE VII Secretion of serotonin of platelets is correlat¬ ed with their aggregation and this reaction was found to be inhibited by 9,12-dioxo-trans-10-octadecenoic acid like aggregation itself. Also this inhibitory effect was found to be dependent on the preincubation 3 period. 100 ^ χJ_ of [ H] serotonin-loaded washed 9 platelets (0.5 x 10 cells) were incubated at 37°C in 400 L of aggregation buffer containing 2mM Ca 2+ acetate. Aggregation was initiated by addition of 3 0.2 units of thrombin and [ H] serotonin release was determined 3 minutes later (mean + SD, n = 4) . The results are shown in Table IV. TABLE III 3 Thrombin Induced Secretion of [ H] Serotonin from Platelets minutes [ H] serontonin test compound pretreatment release (%) none 2 74.3 + 1.3 3 25^ < _M 9,12-dioxo-18:0 2 69.2 + 4.0 2 25 _ 9 ,12-dioxo-tr-18:1 2 29.4 ± 3.7 2 25^1 9,12-dioxo-tr-18:1 5 12.0 ± 1.6 The inhibitory compounds may be administered by the usual pharmaceutically acceptable modes, parenterally by injection or infusion, by ingestion, or the like. The compounds may be administered in amounts between about 0.4 mg to 400 mg per kilogram of body weight-to produce a concentration within the body between about to 1 mM. It is apparent that many modifications and variations of this invention as hereinbefore set forth may be made without departing from the spirit " and scope thereof. The specific embodiments de¬ scribed are given by way of example only and the invention is limited only by the terms of the append¬ ed claims.

Claims: WE CLAIM: 1. A method of inhibiting aggregation of blood platelets in a living body which comprises adminis¬ tering a compound including the structure: [l,4-dioxo-2-ene] . — 2. A method of inhibiting aggregation of blood platelets in a living body which comprises adminis¬ tering a compound of the formula: H wherein R.. and R 2 are aliphatic substituents. 3. A method according to Claim 2 wherein said aliphatic substituents have chain lengths of from 3 to 10 carbon atoms. 4. A method according to Claim 2 wherein said aliphatic substituents are of low polarity. 5. A method according to Claim wherein said aliphatic substituent has at least one added chemical substituent. 6. A method according to Claim 5 wherein said added substituent is selected from the class consist¬ ing of σarboxyl, carboxyl ester and alkoxyl. 7. A method according to Claim 2 wherein said aliphatic substituent is saturated. 8. A method according to Claim 2 wherein at least one of said aliphatic substituents is unsat- urated. 9. A method according to Claim 2 wherein the inhibitory compound is administered in amount suffi¬ cient to establish a concentration in the blood of at least 1 to 6^- . 10. A method according to Claim 9 wherein the inhibitory compound is administered in amount of at least about 0.4 mg per kg of body weight.

Inventors: GELLERMAN JOANEE L, GRAFF GUSTAF, SAND DONALD M, SCHLENK HERMANN, GELLERMAN, JOANEE, L., GRAFF, GUSTAF, SAND, DONALD, M., SCHLENK, HERMANN
Applicants: UNIVERSITY PATENTS INC, UNIVERSITY PATENTS, INC.
Application year: 1985
Main classification: A61K31
IPC codes: A61K31