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United States Patent (19) 11 Patent Number: 4,588,587 Gasic (45) Date of Patent: May 13, 1986 54 METHOD of TREATMENT to INHIBIT Budzynski Et Al.-PSEBM, Vol

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United States Patent (19) 11 Patent Number: 4,588,587 Gasic (45) Date of Patent: May 13, 1986 54 METHOD of TREATMENT to INHIBIT Budzynski Et Al.-PSEBM, Vol United States Patent (19) 11 Patent Number: 4,588,587 Gasic (45) Date of Patent: May 13, 1986 54 METHOD OF TREATMENT TO INHIBIT Budzynski et al.-PSEBM, vol. 168, (1981), pp. 259 & METASTASS 261-265. 75 Inventor: Gabriel J. Gasic, Gulph Mills, Pa. Primary Examiner-Sam Rosen 73) Assignee: Pennsylvania Hospital, Philadelphia, Attorney, Agent, or Firm-Ratner & Prestia Pa. 57 ABSTRACT Method for treating a patient to inhibit metastasis of (21) Appl. No.: 471,197 malignant cells by administering to the patient a thera peutically effective amount of a composition including a 22). Filed: Mar. 1, 1983 blood anticoagulant and a protease inhibitor. A compo 51 Int. Cl............................................... A61K 35/62 sition apparently unique for this purpose is a leech sali vary gland extract, the anticoagulant effect and prote (52) so v. ase inhibitory effect of which are believed to be signifi 58) Field of Search .......................................... 424/95 cant. Other significant components may also be present (56) References Cited in the leech salivary gland extract and these compo nents may act synergistically in producing the observed PUBLICATIONS antimetastatic effect. Gasic et al.-Cancer Research, vol. 43, Apr. 1983, pp. 1633-1636. 11 Claims, No Drawings 4,588,587 1. 2 And some of these agents have shown a marginal (but METHOD OF TREATMENT TO INHIBIT only marginal) capacity to inhibit metastatic activity. METASTASS Notwithstanding these studies, however, there re mains a continuing need for a more effective antimeta BACKGROUND OF THE INVENTION 5 static treatment method. This invention pertains to a method of treatment for It is the general object of the present invention to inhibiting the spread of malignant cells, or metastasis. provide such a method. "Metastasis', as used herein, is defined as the transfer More specifically, an object of this invention is to of malignant tumor cells, or neoplasm, via preformed provide a method of treatment to inhibit metastasis, by body vascular channels (blood vessels or lymphatics), 10 administering a therapeutically effective amount of a or via natural body cavities, usually from the primary composition apparently uniquely adapted to inhibit focus of neoplasia to a distant site in the body, and metastasis. subsequent development of secondary tumors or colo nies in the new location. BRIEF DESCRIPTION OF THE INVENTION Metastasis is an ominous sign of malignancy and often 15 This invention comprises a method for treating an represents a significant factor leading to death. Its treat animal to inhibit metastasis of malignant cells in the ment is difficult to achieve with conventional methods, patient's body by administering to that patient a thera such as surgery and radiotherapy. Efforts to treat cells peutically effective amount of a composition including in metastasis by a systematic therapy, such as the one both a blood anticoagulant and a protease inhibitor or a proposed in this application, have heretofore been 20 largely unsuccessful. specific anticoagulant or protease inhibitor present in a The mechanism by which metastasis occurs is be leech salivary gland extract. Preferably both the blood lieved to involve several steps, such as entry of tumor anticoagulant and protease inhibitor are extracted from cells into the circulation (intravasculation); their trans leech salivary glands. This extract may be produced by port by the blood stream; interactions of circulating 25 freeze drying dissected leech salivary glands, homoge malignant cells with platelets and plasma clotting fac nizing the freeze dried material in an aqueous solvent, tors with activation of the hemostatic system; interac centrifuging the homogenized freeze dried material and tion of the same cells with host cells other than platelets; filtering the centrifuged material to remove particulate arrest of tumor cells, surrounded by platelet aggregates material therefrom. The solvent of choice is water buff and fibrin clots within capillaries; proteolytic attack of 30 ered, for example, by 20 mM/1 Hepes (N-2-Hydrox the blood vessel wall, particularly of its basement mem yethyl Piperazine-N-Ethane Sulfonic Acid) and 10 brane, by tumor enzymes; escape from the circulation mM/1 CaCl2 at a pH on the order of 7.8. system (extravasculation); and formation of secondary The effectiveness of leech salivary gland extract as an tumors or colonies. anti-metastatic agent is believed to be due to a unique Blood sucking animals, such as leeches, mosquitoes 35 and complex combination of anticoagulants, protease and vampire bats have long been known to possess in inhibitors of various kinds, and possibly other constitu their saliva substances which chemically interact with ents in the extract, all of which act additively or syner the blood or blood components. For this reason, leeches gistically on one or several steps of the complex mecha have been used for blood letting since antiquity. Al nism of metastasis. Fractions of this extract, alone or in though this practice achieved great popularity in Eu 40 combination with conventional anticoagulants and pro rope at the beginning of the last century and has since tease inhibitors may also be effective in inhibiting metas been abandoned, interest in the nature and properties of taSS. leech anticoagulants (one of the known components of DETALED DESCRIPTION OF THE the leech saliva which react with blood clotting factors) INVENTION has recently been renewed as a consequence of the 45 greater interest in the biochemistry of blood clotting Leech salivary gland extract, a complex mixture of and the search for new antithrombotic agents. These active compounds, including among others, blood anti investigations have culminated in the isolation and char coagulants and protease inhibitors, has been found to acterization of several leech anticoagulants, such as markedly reduce the number of metastatic tumor colo hirudin, a specific thrombin inhibitor from the species 50 nies induced by intravenous injection of T241 sarcoma Hirudo medicinalis, hementerin, a plasminogen activator cells in mice. from leeches of the species Haementeria lutzi, and he A similar effect has been observed upon injection of mentin, a fibrinogen and fibrin degrading enzyme from certain carcinoma cells. The mechanisms of metastasis leeches of the species Haementeria ghiliani and proba in these animals are believed to be very similar to those bly from leeches of the species Haementeria officinalis. 55 in humans. These tests are indicative that an effective Studies of the salivary glands from Hirudo or Haem method to inhibit the metastatic spread of malignant enteria leeches have also revealed the presence of sev cells in animals may be provided by administering a eral kind of protease inhibitors, i.e., substances capable therapeutically effective amount of leech salivary gland of neutralizing or inhibiting the degrading or digestive extract, or components thereof or combinations of other activity of proteins by conventional enzymes, such as constituents similar thereto. trypsin, plasmin, chymotrypsin, elastase, cathepsins, Tests to date have utilized salivary gland extract, or etc. active fractions thereof, from leeches of Class Hiru With regard to possible use as antimetastatic agents, dinea, Order Rhychobdellida, Family Glossiphonidae conventional anticoagulants, including heparin and and leeches of Class Hirudinea, Order Gnathobdellida, Bothrops atrox venom (Atroxin); antiplatelet agents, 65 Family Hirudinidae, the main prototypes being repre such as aspirin; and inhibitors of proteolytic activity sented, among other leeches, by Haementeria ghiliani from various natural sources, such as leupetin, aproti and Haementeria officinalis, in the first case, and by nin, etc. have all been tested and studied (individually). Hirudo medicinalis, in the second. 4,588,587 3 4. How the leech salivary gland extract acts to inhibit The above conclusions and hypotheses are based on metastasis is not yet fully known. However, the hemo the results of experiments described below, in which are static system has been thought to contribute to metasta used the following materials and methods. sis formation by causing vascular arrest of tumor cells. Tumor It may be hypothesized therefore that the extract's ca 5 pacity to inhibit coagulation, platelet aggregation or Sarcoma T241 (12), a dimethylbenzanthracene both is an important factor in the dramatic antimeta induced tumor originally derived from a C57BL/6 static effect demonstrated. Although T241 sarcoma mouse, was maintained as asciitic tumor in syngeneic cells lack the ability to aggregate platelets in vitro di C57BL/6 mice or B6AF compatible hybrids (Jackson rectly, platelet aggregation may be induced by exposed 10 Laboratory, Bar Harbor, Me). The capacity of the cells collagenous components of the basement membrane of this tumor to induce platelet aggregation and coagul and underlying connective stroma, following attack on lation was assayed in a conventional manner. Addition the vascular lining by T241 sarcoma cells. Applicant of up to 2x 106 T241 cells to 500 ul of heparinized has demonstrated that aggregation of rat platelets in mouse or rat platelet-rich plasma (5-10 units heparin per duced by bovine collagen can be inhibited in vitro by 15 ml) failed to aggregate platelets. The tumor cells, how the salivary gland extract. Therefore, there is a signifi ever, did display a moderate procoagulant activity as cant possibility that the antimetastatic effect of the ex reflected by a shortened
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