|H|||||||||| USOO5278143A United States Patent 19 11 Patent Number: 5,278,143 Shepro Et Al

|H|||||||||| USOO5278143A United States Patent 19 11 Patent Number: 5,278,143 Shepro et al. (45) Date of Patent: Jan. 11, 1994

(54) PROPHYLACTIC AND THERAPEUTIC OTHER PUBLICATIONS METHODS FOR TREATING NTER LEUKIN-MEDIATED EDEMAS Rudinger, Peptide Hormones, Parsons (Ed.) U. Park t Press, Baltimore, pp. 1-7 (1976). (75) Inventors: David Shepro, Boston, Mass.; J. Doukas et al., Blood, vol. 69, No. 6 pp. 1563-1569 (Jun. Steven Alexander, Nashville, Tenn. 1987). Frimmer, Chem. Abstracts, vol. 71, No. 99937m (1969). 73) Assignee: Trustees of Boston University, Wieland et al., Crit. Rev. Biochem. vol. 5, pp. 185-260 Boston, Mass. (1978). (21) Appl. No.: 807,668 Welbournet al., J. Appl. Physiol 70: 1364-1368 (1991). 22 illed: Dec. 16, 1991 Primary Examiner-Y. Christina Chan 22 Filed ec. 10, Attorney, Agent, or Firm-David Prashker Related U.S. Application Data 57 ABSTRACT m 63 continuation of ser. No. 416,905, Oct. 4, 1989, aban- Unique methods for treating interleukin-mediated ede doned, which is a continuation-in-part of Ser. No. mas in living subjects are provided comprising adminis 47,121, Oct. 4, 1989, abandoned, and a continuation- tering an effective amount of a composition selected in-part of Ser. No. 185,650, Apr. 25, 1988, abandoned. from the group consisting of phallotoxins, phallotoxin analogues, antamanide, or an antananide analogue to 51 Int. Cl...... A61K 37/02; CK /6 the subject. The methods offer prophylactic and thera 52 576.5/35567; 17,353 peutic modes of treatment for both localized and sys s s 8 temic interleukin-mediated edemas. The compositions 58) Field of Search ...... 530/317,321; 514/9, of choice may be applied topically or given parenter 514/11, 870, 922; 424/85.2 ally; and may be employed with other diverse agents for (56) References Cited treatment of both inflammatory and non-inflammatory edemas. U.S. PATENT DOCUMENTS 4,690,915 9/1987 Rosenberg ...... 514/2 7 Claims, 3 Drawing Sheets U.S. Patent Jan. 11, 1994 Sheet 1 of 3 5,278,143

** 3: SIG. OIFF. FROM CONTROL SALINE (P<0.5, P<0.0) RESPECTIVELY ) ava SIG. DIFF. FROM IL-2 106 U/M 1 (P<0.05, PCO,O RESPECTIVELY)

E at m - - a de e C C UMd a UM (V) MD GMD N. a. N N N a N. Cn N. cn (N ad - - ON t C l - - ad 3 - A. a with P O

sed - n - a 5 MD Mo CM) N EA. S SQ s -N Nup CN NN CN and a 22 - b A. a es Ee S2 U.S. Patent Jan. 11, 1994 Sheet 2 of 3 5,278,143

3,3:3: SIG. DIFF. FRON CONTROL SALINE (P<0.05, P & O. O. RESPECTIVELY ) -- ~ SIC. DIFF. FRON IL-2 IO 6U/M (P<0.05, PCOO RESPECTIVELY

i FIG. 3

i F G. 4. U.S. Patent Jan. 11, 1994 Sheet 3 of 3 5,278,143

%,3+ SIG. DIFF. FROM CONTROL SALINE (P<0.05, PCOO RESPECTIVELY ) ava SIG. Diff. FROM IL-20 6U/M (P<0.05, P&O.O RESPECTIVELY ) 5 a r

2250 2000 750 500 250 000 d ae CSS C S CC C C C CC S.C 750 C C S. O C C C C3 CC

SS C 500 O C C

SSC C 250 C 33. C 3.

3. SALNE SNE pRobinS SSs froE CONTROL IL-2 L-2 IL-2 FIG.6 TREATMENT 5,278,143 1 2 ter "nm') in radius or by slits about 8 nm wide. There is PROPHYLACTIC AND THERAPEUTICMETHODS also believed to be a system of larger sized pores about FOR TREATING INTERLEUKIN-MEDIATED 25 nm in radius which accounts for the small quantities EDEMAS of protein and other large solutes that normally cross the endothelial wall barrier. GOVERNMENTAL SUPPORT A variety of different disturbances can induce a con Research support for the present invention was pro dition of edema. These include: an elevated venous vided by a grant from the Community Technology hydrostatic pressure which may be caused by thrombo Foundation of Boston, University. sis of a vein or any other venous obstruction; hypopro This application is a continuation of application Ser. 10 teinemia with reduced plasma oncotic pressure result No. 416,905, filed Oct. 4, 1989, now abandoned; which ing from either inadequate synthesis or increased loss of is a continuation-in-part of U.S. patent application Ser. albumin; increased osmotic pressure of the interstitial No. 185,650 filed Apr. 25, 1988 now abandoned; and a fluid due to abnormal accumulation of sodium in the continuation-in-part of copending U.S. patent applica body because renal excretion of sodium cannot keep tion Ser. No. 417,121, filed Oct. 4, 1989, now aban 5 pace with the intake; failure of the lymphatics to re doned. move fluid and protein adequately from the interstitial space; an increased capillary permeability to fluids and FIELD OF THE INVENTION proteins as occurs in the inflammatory response to tissue The present invention is concerned with inflamma injury; an increased mucopolysaccharide content tory and non-inflammatory (hydraulic) associated ede 20 within the interstitial spaces; and an iatrogennic induced mas and is particularly directed to prophylactic and or mediated increase or accumulation of fluid and pro therapeutic methods for treating localized and systemic tein resulting from the administration of a pharmacolog edemas caused or mediated by interleukins. ically active lymphokine during the course of treatment BACKGROUND OF THE INVENTION by a physician or surgeon. This last-identified distur 25 bance merits a more detailed examination in view of Edema is the term generally used to describe the recent developments in immunotherapy. accumulation of excess fluid in the intercellular (inter Lymphokines comprise a broad class of biologically stitial) tissue spaces or body cavities. Edema may occur active substances which are secreted by various types of as a localized phenomenon such as the swelling of a leg lymphocytes in-vivo and in-vitro, especially by differ. when the venous outflow is obstructed; or it may be 30 ent populations of T-cell lymphocytes. Perhaps the systemic as in congestive heart failure or renal failure. When edema is severe and generalized, there is diffuse most controversial presently is the family of interleu swelling of all tissues and organs in the body and partic kins, which presently comprises: Interleukin-1 (“IL-1") ularly pronounced areas are given their own individual involved in the activation of resting T-cells; Interleukin names. For example, collection of edema in the perito 35 2 ("IL-2') which mediates the proliferation of T-cells neal cavity is known as "ascites'; accumulations of fluid and induces cytotoxic activity in T-cells; Interleukin-3 in the pleural cavity are termed "hydrothorax'; and ("IL-3') which causes the proliferation of mast cells edema of the pericardial sac is termed "pericardial effu and granulocytes; Interleukin-4 ("IL-4') which medi sion' or "hydropericardium'. Non-inflammatory ates the proliferation, activation, and differentiation of edema fluid such as accumulates in heart failure and B-cells, T-cells, and natural killer cells; and Interleukin renal disease is protein poor and referred to as a "transu 6 ("IL-6”) which induces the growth and differentiation date'. In contrast, inflammatory edema related to in of B-cells and T-cells Spits et al., J. Immunol. 139:1142 creased endothelial permeability is protein rich and is (1987); Kawakami et al., J. Exp. Med 168:2183 (1988); caused by the escape of plasma proteins (principally Spits et al., J. Immunol. 141:29 (1988); and Tartakovsky albumin) and polymorphonuclear leukocytes (hereinaf 45 et al., J. Immunol. 141:3863 (1983)). ter "PMNs') to form an exudate. Of particular interest is Interleukin-2 ("IL-2') and its Edena, whether inflammatory or non-inflammatory analogues whose isolation, chemical formulation and in nature, is thus an abnormality in the fluid balance structure, and synthesis by conventional wet chemis within the microcirculation which includes the small tries and by recombinant DNA techniques have been arterioles, capillaries, and post-capillary venules of the 50 intensively pursued see for example U.S. Pat. Nos. circulatory system. Normal fluid balance and exchange 4,490,289; 4,138,927; 4,569,790; 4,578,335; 4,761,375; is critically dependent on the presence of an intact and 4,518,584; 4,604,377; 4,564,593; and the references cited metabolically active endothelium within the vascula therein). The use of Interleukin-2 as a therapeutic com ture. Normal endothelium is a thin, squamous epit position has followed two very different approaches. belium adapted to permit free, rapid exchange of water 55 The first line of development is exemplified by the IL-2 and small molecules between plasma and interstitium; immunotherapy technique for treating cancers origi but one which limits the passage of plasma proteins with nated by Dr. Steven A. Rosenberg Rosenberg, S.A., increases in protein size. Immuno. Today 9:58-62 (1988) and the references cited The endothelial lining of all arterioles and venules, therein). That approach involves the removal of lym and most capillaries in the body, is of the continuous phoid cells from a tumor-bearing host; an in-vitro ex type, having an unbroken cytoplasmic layer with pansion of the host's cells by culture in Interleukin-2 closely apposed intercellular junctions. Physiological containing culture media to produce lymphokine studies Renkin, E., Circ. Res. 41:735-743 (1977); Ren activated killer ("LAK') cells; and a re-introduction of kin, E., ACTA Physiol. Scand. (Suppl.) 463:81 (1979); the LAK cell culture into the living host accompanied Bottaro et al., Microvasc. Res. 32:389-398 (1986) have 65 by successive administrations of IL-2 directly to the demonstrated normal endothelial permeability for tumor-bearing host. This approach has been termed water and small molecules by the existence of water "adoptive immunotherapy." A variation of this devel filled small pores approximately 6 nanometers (hereinaf opment is the use of IL-2 directly as an anti-cancer 5,278,143 3 4. agent. This is exemplified by Patent Nos. WO-8600334 muscularly. Phalloidin acts by binding actin, a cytoske published 8601 16; JP-60185721 published 850921; U.S. letal protein Russo et al. Am. J. Pathol. 109:133 (1982). Pat. No. 4,645,830; EP-145390 published 850619; and Reviews of the chemical structure and toxicology of all WO8500606 published 850214. toxins derivable from Amanita phaloides including The second general approach has been to chemically phalloidin have been reported in the literature Wieland link the Interleukin-2 molecule to another ligand having and Wieland, Pharmacol. Rey, 1:87-107 (1959); Wie known biological properties to form a hybrid. The IL-2 land, T., Fortschr. Chem. Org. Naturst. 25:214-250 portion of the hybrid serves a specific binding protein (1967); Faulstich H. and T. Wieland, Eur, J. Biochem. able to selectively bind to IL-2 receptor sites on the 22:79 (1971); Wieland and Faulstich, Crit. Rev. Biochem. surface of living cells; the IL-2 portion of the hybrid 10 5:185-260 (1978)). thus served as the means of delivering and selectively Sporadically, some investigators have employed introducing the other component of the hybrid into a phalloidin within in-vitro experiments for its effects chosen cell population having IL-2 receptor sites on upon living cells. For example, the interaction of phal their cell surface. This second approach is exemplified loidin with actin was one of the earliest reported dem by: U.S. Pat. Nos. 4,675,382 and 4,745,180, GB-2189393 15 onstrations Lengsfeld, A. M., Proc. Natl. Acad. Sci published 871028; EP-269455 published 88060; EP USA 71:2803-2807 (1974). Subsequently, it was shown 256714 published 880224; and EP236987 published that phalloidin treatment induces changes in the inter 870916. cellular junctions of rathepatocytes Montsano et al., J. A major problem and deficiency for the in-vivo use of Cell. Biol. 67:310-319 (1975)). More recently, phalloidin interleukins generally and of IL-2 in particular has been 20 was employed to increase the resistance of Necturus the now well recognized phenomenon of uncontrolled gallbladder epithelium to the passage of an electrical edema as a concomitant side-effect of interleukin admin current Bentzel et al., Amer. J. Physiol. 239:C75-C89 istration. The observation of massive edema locally and (1980). The use of phalloidin in such investigative stud systemically has been termed "vacular leakage syn ies has therefore been primarily as a research tool by drome' and is now seen as a regular and unavoidable 25 which to further characterize and elucidate the mecha consequence of using interleukins therapeutically in nism of cytoskeleton action within living cells. either uncoupled or complexed/hybridized form. Rep The other major class was identified and founded resentative publications describing the uncontrolled based on the one naturally occurring substance, antama edemas caused or mediated by the administration of an nide, which was not only completely non-toxic of itself; interleukin include: Carlsen, E. and H. Prydz, Thrombo 30 but also was found capable of actually annulling the sis and Harmostasis 58:257 (1987); Giddings, J. C. and L. toxic effects of fatal doses of phalloidin and/or of pro Shall, Thrombosis and Haemostasis 58:31 (1987); Kota tecting the liver completely when administered in ther sek et al., Clin. Res. 35:660A (1987); Ellison et al., Anat. apeutic doses Wieland et al., Angew. Chem. 80:208 Rec. 218:41A (1987); and the references cited therein). (1968). Subsequent investigations of this cyclic deca In so far as is presently known, there is no effective 35 peptide, antamanide, then proceeded in two different composition or method to meaningfully control, re directions: one research effort involved methods of duce, or substantially eliminate the resulting vascular synthesizing, purifying, and preparing analogues of leakages and the massive edemas caused and/or medi antamanide. These investigations are examplified by: ated by the in-vivo administration of an interleukin for U.S. Pat. Nos. 3,705,887 and 3,793,304; Anderson et al., any therapeutic purpose. Furthermore, there is pres J. Am. Chem. Soc. 88:1338-1339 (1966); Anderson et al., ently no effective agent or admixture of substances J. Am. Chem. Soc. 89:5012-5017 (1967); Wieland, T., useful as a prophylactic against an interleukin-mediated Angew. Chem. (Internat. Edit) 7:204-208 (1968); Ov edema. chinnikov et al., Proc. Eur, Pept, Synp. 11:403-415 Remote from and completely unrelated to these in (1973); Wieland et al., Liebig's Ann, Chen., number 3, vestigations of interleukins were other research efforts 45 pages 371-380, 1977; Burgermeister et al., Eur, J. Bio directed towards the isolation and identification of the chem. 44:305-310 (1974); Tonelli, A. E., Biochemistry component substances of the poisonous green fungus 12:689-692 (1973); Patel, D.J., Biochemistry 12:677-688 Amanita phaloides, known as the "green death cap' or (1973); Ivanov et al., Biochen. Biophys. Res. Conn. "deadly agaric' mushroom Lynen and Wieland, Justus 42:654–663 (1971); and Bir et al., J. Peptide Protein Res. Liebigs, Ann. Chem. 533:93-117 (1938); Wieland and 50 13:287-295 (1979)). Schnabel, Justus Liebigs Ann. Chem, 657: (1962). At In comparison, the other investigations focused upon least ten peptide-like substances of complex structure the physiological and pharmacological attributes of have been identified; most of these substances have naturally occurring antamanide and its synthetic ana proven to be extremely toxic liver toxins Liebig's Ann. logues. These investigations are exemplified by the foll Chem., volume 617, page 152, 1958; Pharmacol Reviews, 55 lowing: Faulstich et al., Hoppe Scylers. Z. Physiol. Chem. volume 7, page 87, 1959; Liebig's Ann. Chem., volume 359:1162-1163 (1974); Ovchennikov et al., Experientia 704, page 226, 1967). Upon isolation and empirical anal 28:399-401 (1972); Wieland et al., Proc. Nat Acad. Sci. ysis of the naturally occurring individual components of U.S.A. 81:5232-5236 (1984); Carle, I. L., Proc. Nat. Amanita phaloides, however, investigators found at Acad. Sci. U.S.A. 82:7155-7159 (1985); Munter, K. D. least two different naturally occurring classes of chemi 60 and H. Faulstich, Biochim. Biophys. Acta 860:91-98 cal compositions: phallotoxins and antamanide. (1986); Nielsen, O., Acta Pharmacol. Toxicol. 59:249-251 The class of phallotoxins as a whole is best exempli (1986); and Raymond et al., Eur, J. Pharmacol. fied by the substance known as phalloidin, one of seven 138:21-27 (1987). naturally occurring member toxins forming the class. In all of these published investigations and reports, Phalloidin is very rapid in action. High dose levels 65 the cyclic decapeptides comprising antamanide and its given intramuscularly cause death of mice or rats within analogues were recognized solely as chemical agents one or two hours. The LD50 dose in albino mice is only capable at very low dosage of counteracting the effects 3.3 micrograms per gram of body weight given intra of an absolutely fatal dose of phalloidin or of com 5,278,143 5 6 pletely protecting the liver against such a fatal dose of FIG. 6 is a graph illustrating the decrease in broncho phalloidin. Only recently was there any investigation alveolar lavage fluid protein after in-vivo administra into areas concerning cell proliferation and wound heal tion of Interleukin-2, phalloidin, and Interleukin-2, and ing Choi et al., FASEB J. 3:A290, Abstract No. 368 antamanide and Interleukin-2 respectively. (1989)). This Abstract was the first publication to sug 5 gest that antamanide might serve as a therapeutic agent DETALED DESCRIPTION OF THE in the treatment of vascular disease. PREFERRED EMBODIMENTS Accordingly, the use of antamanide and the synthetic The present invention is the general methodology for analogues remains primarily and predominantly as an prophylactically or therapeutically treating localized or anti-toxin against the effects of phalloidin; and phal O systemic interleukin-mediated or induced edemas in a lotoxins as a class are known primarily for their toxic living subject which comprises the step of administering effects and, therefore, used primarily as a research tool. an effective amount of an antamanide, or an antamanide There is, thus, no known relationship for these sub analogue, or a phallotoxin, or a phallotoxin analogue to stances with respect to controlling interleukin-mediated the living subject either before or after ocurrence of the edemas in-vivo. 15 interleukin-mediated edema in the subject. The broadest chemical definition of the class of compositions con SUMMARY OF THE INVENTION prising antamanides and its analogues is provided by The present invention provides methods for thera Formula I below: peutically or prophylactically treating interleukin mediated edema in a living subject. The method for 20 therapeutically treating interleukin-mediated edema (I) comprises the step of administering an effective amount of a substance selected from the group consisting of phallotoxins, phaliotoxin analogues, antamanide, or an antamanide analogue to the subject after occurrence of the edema. The method for prophylactically treating interleukin-mediated edema comprises the step of ad ministering an effective amount of a phallotoxin, a phal lotoxin analogue, antananide, or an antananide ana 30 wherein Windividually is an amino acid having at least logue to the subject prior to the occurrence of an inter one ring structure comprised of not more than 6 carbon leukin-mediated edema. Either methodology inhibits atoms; the permeability of fluid, macromolecules, and blood X individually is an acyclic amino acid comprised of cells across the microvasculature thereby acting di 3-9 carbon atoms; rectly on the clinical manifestations of the edema and 35 Y individually may be omitted entirely, but when avoiding indirect metabolic cascades and pathways. present is selected from the group consisting of hydro The prophylactic methodology can be employed in gen, a hydroxyl group, a halogen, a hydrocarbon, and a settings where iatrogenic-induced edema typically oc substituted hydrocarbon; and curs such as with the use of adoptive immunotherapy or Z individually may be omitted entirely, but when the use of interleukin-containing hybrids. The therapeu present is selected from the group consisting of hydro tic methodology can be used to attenuate both an in gen, a halogen, a hydrocarbon, and a substituted hydro flammatory response as well as a non-inflammatory carbon. reaction current with the in-vivo administration of an A preferred broad definition of the cyclic decapep interleukin. Moreover, the antamanides, antamanide tide compositions and structures comprising the class of analogues, phallotoxins, and phallotoxin analogues may 45 be used alone or in combination with other substances antamanides and antamanide analogues is given by For known to affect cellular metabolic pathways. Inula II below: BRIEF DESCRIPTION OF THE FIGURES (II) The present invention may be more easily and com 50 pletely understood when taken in conjunction with the accompanying drawing, in which: FIG. 1 is a graph illustrating the spleen wet to dry weight ratios after in-vivo administration of Interleukin 2, phalloidin, and antamanide alone and in combination; 55 FIG. 2 is a graph illustrating the kidney wet to dry weight ratios after in-vivo administration of Interleukin 2, phalloidin, and antamanide alone and in combination; wherein W individually is an amino acid having a ben FIG. 3 is a graph illustrating the liver wet to dry zene ring in its structure; weight ratios after in-vivo administration of Interleukin X individually is an amino acid selected from the 2, phalloidin, and antamanide alone and in combination; group consisting of valine, alanine, leucine, and isoleu FIG. 4 is a graph illustrating the heart wet to dry cine; weight ratios after in-vivo administration of Interleukin Y individually may be omitted entirely, but when 2, phalloidin, and antamanide alone and in combination; present is selected from the group consisting of hydro FIG. 5 is a graph illustrating the lung wet to dry 65 gen, a hydroxyl group, a halogen, a hydrocarbon moi weight ratios after in-vivo administration of Interleukin ety, and a substituted hydrocarbon moiety; and 2, phalloidin, and antamanide alone and in combination; Z individually may be omitted entirely, but when and present is selected from the group consisting of hydro 5,278,143 7 8 gen, a halogen, a hydrocarbon moiety, and a substituted Within the various embodiments encompassed by hydrocarbon moiety. Formulas I-IV inclusively, it will be recognized that the It will be recognized and appreciated that Formulas I amino acids at the No. 1 position, typically L-valine, and II respectively are presented using conventional and at the No. 4 position, typically alanine, may be chemical structure, format, and notations for amino 5 substituted by other amino acids. At a minimum, these acids and peptide organization as those found in Albert other amino acids are acyclic-and by definition do not L. Lehninger's text, Biochemistry, The Molecular Basis of contain any ring structure within their composition. In Cell Structure And Function, 2nd edition, Worth Pub addition, it is preferable that these amino acids also do lishers, Inc., 1977-the text of which is expressly incor not comprise a secondary nitrogen atom, a secondary porated by reference herein. Moreover, Formulas I and 10 carboxylic acid group, nor any sulfur atom. The pre II respectively by their definitions intend that all pres ferred choices of "X' include L-leucine and L-isoleu ently known and future embodiments of naturally oc cine in addition to L-valine and L-alanine. curring and non-naturally generated substances-which In comparison, the suitable amino acids at position are by chemical formulation and structure one of those Nos. 5, 6, 9, and 10 within the cyclic decapeptide struc cyclic decapeptides forming the class of antamanides 15 ture each require the presence of a cyclic ring compo and antamanide analogues (including all substituted and nent. Preferred are hydrophobic side-chains such as a derivatized forms)-lie within the scope of the present benzene ring of 6 carbon atoms. However, it is expected invention. However, the more desirable embodiments that other ring configurations ranging from 3-6 carbon are those described and synthesized by U.S. Pat. Nos. atoms which are alternatively saturated or unsaturated 3,705,887, 3,793,304, and 3,211,716-the texts of each 20 in varying degree will also be useful. Accordingly, the being expressly incorporated by reference herein. These broadest definition provided by Formula I requires the issued patents not only provide the best embodiments of "W" positions only to be filled by an amino acid having naturally occurring and non-naturally generated an at least one ring structure comprised of not more than 6 tamanides; but also provide complete and detailed pro carbon atoms. cedures and techniques for synthesizing and purifying 25 The given recitations of "Y" and “Z” identify com antamanides and antamanide analogues for use in the monly available chemical substitutions and derivatized present methodologies. forms for the general membership of the class compris Accordingly, a most preferred embodiment use for ing antamanides and antamanide analogues. The various treatment of interleukin-mediated edema is the naturally substitutions and additions to the cyclic decapeptide occurring antamanide defined by Formula III, which is: 30 structure are performed using commonly known syn theses and chemical reaction techniques. All of the embodiments of "Y" and “Z” are matters of personal (III) choice and convenience well within the skills of the ordinary practitioners in this field. The preferred meth Pro8 35 ods for synthesizing the cyclic decapeptides comprising Pro7 Phes Formulas I-IV inclusive are those conventionally known and described in detail within U.S. Pat. Nos. 3,705,887, 3,793,304, and 3,211,716 respectively. The Other preferred embodiments of non-naturally occur synthesis is preferrably performed using acyclic deca ring antamanides believed useful for treating interleu peptides which are then cyclized into the overall cyclic kin-mediated edemas is given by Formulas IVA-IVD: form using conventional methods in peptide synthesis. In general, the cyclization from linear into cyclic form may take place at any position using the acyclic precur Tyr9 Vall (IVA) sor peptide unit where an amino group and a carboxyl P / N Phel0 / N r group are available for reaction. In the case of the Pro7 Phes Pro amino acid proline, the imino group (HN=) may be N / N / present at the end of the chain instead of an amino Phe6 Ala group (H2N-). 50 As noted in the cited patents, two processes are Phe9 Vall (IVB) mainly involved, both of which however are suitable P / N Tyrl / N P for general application and synthesis. In the first pro Pro7 Phes Pro cess, one end of the acyclic chain, preferrably the car N / N / boxyl group, is applied in an activated form, while the Phe6 Ala 55 amino reactive group has already been reversibly pro tected, or if this is not the case, must initially be pro Phe9 Vall (IVC) tected. The amino protective group is then selectively P / N Phel / N P split off in such a way that the amino group thus liber Pro7 Tyrs Pro ated is protected simultaneously by proteination. The N / N / deproteination of the reactive amino group by bases in Phe6 Ala highly dilute solution leads to cyclization. The second general process employs a peptide zwit Phe9 Vall (IVD) terion, the amino group of which is already present in P M. N. Phel0 / N r proteinated condition. By activation of the carboxyl Pro7 Phes Pro 65 group and the addition of a base in diluted solution, or N / N / by addition of a dehydrating agent to the peptide zwit Tyré Ala terion, the cyclic end-product is also obtained in one step. Both of the general cyclization methods are per 5,278,143 10 formed in relatively great dilutions in order to suppress di- and poly-condensations. TABLE I-continued In comparison, phallotoxins and phallotoxin ana PHALLOTOXIN SIDE CHANS logues are bicyclic heptapeptides and are most broadly split Iw Seco-8-Phallotoxin R-R as in If, but defined by Formula V and Table 1 below. 5 instead of peptide bond at PCOOiBut at N and NH2 at Co Ix Bis-seco-15,18-phallotoxin Open at P and between H3C-CH-CO-NH-CH-CO-NH --al threonine and cysteine Source: CH2 f O Faustich, H. and T. Wieland, Eur, J. Biochem. 22:79-86 (1971). NH NH The preferred embodiments of phallotoxins as a class N are the naturally occurring compounds isolated from S N Amanita phaloides and include the bicyclic heptapep CO cf.2 R5 cH-R2 15 tides phalloidin, phallacidin, phalloin, phallisin, pro phalloin, phallisacin, and phallacin. The composition Na-CO- th and individual methods of preparation for each natu R4 NH-CO-CH-NH-CO rally occurring member are conventionally known and R3 20 published in the scientific literature Wieland et al., Pharmacol. Rey 11:87-107 (1959); Wieland et al., Crit. Rey, Biochem, 5:185-260 (1978)). Methods for generat TABLE I ing the chemically synthesized phallotoxin analogues of PHALLOTOXIN SIDE CHANS Formula V and Table I are conventionally known. A. Natural Phallotoxins 25 Each member within the class phallotoxins is be a Phalloidin R = C(CH3)OH-CH2OH lieved to be individually active and effective in varying R3 = CH3 degree; and each is expected to act directly on the vas R4 = C(H)OH-CH3(threo) culature of the cell and not via an indirect metabolic R = OH(allo) R = H b Phaloin R = C(CH3)OH-CH3 cascade for control of interleukin-mediated edemas. R2 = R as in Ia 30 Within the class, phalloidin is presently considered to be ic Phallisin R = C(OH) (CH2OH)2 the most effective composition; and for this reason, the R2 = R as in Ia remainder of the detailed description will focus upon Id Phalacidin R = C(CH3)OH-CH2OH R2 = CH(CH3)2 and utilize phalloidin alone or in combination with an 3 = C(H)OH-CO2H(erythro) tamanide as the best representatives of their respective 4 = OH(allo) R5 = H 35 classes. It will be expressly understood, however, that B. Chemically Modified any of the other members within the class consisting of Phallotoxins antananide, antamanide analogues, phallotoxins, and ie Ketophalloidin R = CO-CH3 R2 = R as in Ia phallotoxin analogues may be similarly employed and If Norphalloin R = CH2-CH3 used effectively as needed or desired. R2 = R as in Ia 40 In-vivo treatment with antamanides, or antamanide g Monoacetylphalloidin R = C(CH3)OH-CH2OAc analogues, or phallotoxins, or phallotoxin analogues is R2 = R as in Ia Ih Monotosylphalloidin R = C(CH3)OH-CH2-Otos intended to be a general methodology for treatment of R2 = R as in la interleukin-mediated edemas in living subjects, particu Ii Diacetylphalloidin I R = as Ig larly humans. The scope of effective treatment using the R2 = as in Ia 45 R* = C(H) OAc-CH3 present invention includes: both prophylactic and thera R = R5 as in Ia peutic applications; treatment of localized or systemic Ij Diacetylphaloidin II R = as in Ig interleukin caused edemas; and treatment given inde R2 = R as in Ia pendently or in combination with other medical and/or R = OAc(allo) R = H Ik Triacetylphalloidin R = R as in Ii surgical modalities. The present invention is useful and R = R as in Ig 50 effective with any one or any combination of these Il Ditosylphalloidin R = as in Ih, second parameters. tosyl either in Ror Any of the antamanides, or antamanide analogues, or Rof Ih Im Tritosylphaioidin R = as in Ih, 2nd and phallotoxins, or phallotoxin analogues encompassed by 3rd tosyl in R and Formulas I-V respectively may be administered to the R of Ih 55 living subject by one of two different routes: topically In Tribomophalloidin 3 Br instead of OTos in by direct application to the skin of the subject; and Im (probably Walden inversion in R and parenterally by injection or perfusion. If the substance R) of choice is to be applied topically, the heptapeptide or Io N-Methylphalloidin R* = CH3 in Ia decapeptide composition can be admixed in a pharma Ip N-Ethylphalloidin R = CHs in la 60 cologically inert topical carrier such as a gel, an oint Iq N-Propylphalloidin R = n-C3H7 in a Ir N-Pentylphalloidin R = n-C5H11 in Ia ment, a lotion, or a cream; and includes such carriers as Is N-tert. Butylphalloidin R = t-C4H9 in la water, glycerol, alcohol, propylene glycol, fatty alco It carbamoylmethylphailoidin R = CH2CONH2 in Ia hols, triglycerides, fatty acid esters, or mineral oils. C. Ring Open Products Other possible topical carriers include liquid petrola Fu Dothiophalloidin in P of Ia S removed, 65 tum, isopropylpalmitate, polyethylene glycol, ethanol, substituted by 2 H at bridgebends polyoxyethylene monolourate in water, sodium lauryl v Seco-15-Phaloidin Peptide bond in P of a sulfate in water, and the like. Other materials such as Ia hydrolytically anti-oxidantts, humectants, viscosity stabilizers, and the 5,278,143 11 12 like may also be added as desired or necessary. In addi tion, it is expected (and in many instances desirable) that EXPERIMENTAL SERIES the substance of choice be disposed within devices Protocol: In-Vivo placed upon, in, or under the skin; such devices include 1. Assay Models: Vascular leak syndrome is induced patches and implants which release the active material 5 rapidly in adult, male rats (500 g) by a continuous infu into the skin or body either by diffusion or by an active sion for 1 h of 106 units of IL-2 (n=36). Five hours later release mechanism. the animals are killed and edema is quantitated by nea Alternatively, if the antananide, or antananide ana suring wet to dry (W/D) ratios for the lungs, kidneys, logue, or phallotoxin, or phallotoxin analogue is to be liver, heart, and spleen. given parenterally, it is expected that the composition O 2. Controls: Animals are pretreated with 0.5 ml saline for 30 min. prior to the IL-2 infusion (n=28). will be prepared in sterile form; in multiple or single 3. Experimental Animals: Each animal is pretreated dose formats; and dispersed in a fluid carrier such as with either Phallin A (antamanide) or Phallin B (phal sterile physiological saline or 5% dextrose solutions loidin), 20 ug/0.5 ml saline as a single bolus 30 min. commonly used with injectables. 15 prior to the infusion of IL-2 (n=24). Each animal then In general, the concentration of the chosen antama receives at 30 min. intervals a booster injection of 20 ug nide or antananide analogue (employed independently of Phallin A or Phallin B for the duration (6 h) of the or combined with other substances) which may be ef experiments. fectively employed for prophylactic and/or therapeutic 4. Protein Analysis: Samples obtained by bronchoal treatment of living subjects is expected to be: for topical 20 veolar lavage are quantitated for protein concentration applications, a range concentration from about 1-10 as another means of determining the relative loss of micrograms (hereinafter "ug") per gram of topical car barrier integrity in the experimental animals compared rier. For intravenous, perfusion and other parenteral to controls. administration, a concentration range of from about 1.0 micromolar-0.1 micromolar per Ll of blood. 25 Results Similarly, the concentration of phallotoxin or phal Spleen Wet to Dry Ratio: As shown by FIG. 1, ad lotoxin analogue (employed independently or combined ministration of phalloidin (1 uM) alone, antamanide (1 with other substances) which may be effectively en uM) alone, or 105 U/ml Interleukin-2 alone did not ployed for prophylactic and/or therapeutic treatment 30 produce any significant splenic edema. 106 U/ml IL-2 of living subjects is expected to be: for topical applica also failed to induce splenic edema, which remained tions, a range concentration from about 1-10 micro unmodified by antamanide or phalloidin. grams (hereinafter "ug') per gram of topical carrier. Kidney Wet to Dry Ratio: As seen in FIG. 2, admin istration of phalloidin (1 uM) alone, antamanide (1 um) For intravenous, perfusion and other parenteral admin alone, or 105 U/1 Interleukin-2 alone did not produce istration, a concentration range of from about 1.0 mi 35 any significant renal edema. Administration of 10°U/ml cromolar-0.1 micromolar per Li of blood. IL-2 produced a significant increase in edema, which In addition, when the chosen phallotoxin heptapep was significantly (p<0.05) worsened by phalloidin (1 tide or antamanide decapeptide composition is em uM). This edema was signnificantly improved (p<0.05) ployed for prophylactic or therapeutic treatment of an by antamanide (1 uM). interleukin-induced or involved edema which is a coin Liver Wet to Dry Ratio: As illustrated by FIG. 3, cidental characteristic of an inflammatory reaction, it is administration of phalloidin (1 uM) alone, antamanide most desirable that the heptapeptide or decapeptide be (1 uM) alone, or 105 U/ml Interleukin-2 alone did not administered in admixture with a variety of anti-inflam produce any significant hepatic edema. Administration matory compounds. These include: aspirin, ibuprofen, 45 of 106 U/ml IL-2 produced no significant increase in thromboxane synthase inhibitors; receptor antagonists edema. IL-2 plus phalloidin (1 uM) produced significant for the thromboxanes; prostanoid metabolic drugs; ste (p<0.05) edema. No significant change in wet/dry roids; and superoxide and free radical scavengers. weight ratio was seen in an antamanide/IL-2 co-treat A major advantage of the present invention is its ment. ability to prophylactically or therapeutically treat inter 50 Heart Wet to Dry Weight Ratio: As provided by leukin-mediated edema, either at localized sites or sys FIG. 4, administration of phalloidin (1 uM) alone, an temically. To empirically demonstrate the clinical cir tamanide (1 uM) alone, or 10 U/ml Interleukin-2 alone cumstances in which the present methodology can be did not produce any significant cardiac edema. Admin beneficially employed, a single cyclic decapeptide de istration of 106 U/mml IL-2 produced a significant 55 (p<0.01) increase in edema, which was significantly fined by Formula III above was empirically evaluated. reduced by either phalloidin (1 uM) (p<0.01) co-treat The embodiment is the naturally occurring form pro ment, or antamanide co-treatment (1 uM) (p<0.01). duced and isolatable from the Amanita phalloides fun Lung Wet to Dry Weight Ratio: As presented by gus. Its amino acid sequence comprises L-valine at the FIG. 5, administration of phalloidin (1 uM) alone, an No. 1 position; L-alanine at the No. 4 position; L tamanide (1 uM) alone, or 10 U/ml Interleukin-2 alone phenylalanine at position Nos. 5, 6, 9, and 10; and L did not produce any significant pulmonary edema. Ad proline at position Nos. 2, 3, 7, and 8. For ease of de ministration of 106 U/ml IL-2 produced a significant scription annd for clarity of presenntation, this embodi (p<0.01) increase in edema, which was significantly ment will be termed "antamanide.' Similarly, a single reduced by either phalloidin (1 uM) (p<0.05) co-treat bicyclic heptapeptide defined by Formula V was empir 65 ment, or antamanide co-treatment (1 uM) (p<0.05). ically tested. This embodiment was phalloidin which Protein Concentrations of Alveolar Fluid: As pres also is a naturally occurring form isolatable from the ented by FIG. 6, the administration of phalloidin (1 uM) same fungus. and 106 U/ml Interleukin-2 in admixture or the adminis 5,278,143 13 14 tration of antamanide (1 uM) and 106 U/ml Interleukin Z individually is omittable entirely but when present 2 in combination drastically reduced the accumulation is selected from the group consisting of hydrogen, and concentration of protein in bronchoalveolar lavage a halogen, and a hydrocarbon moiety. fluid when compared to the effects of administering 106 2. A method for prophylactically treating an interleu U/ml Interleukin-2 alone. The degree of difference is 5 kin 2-mediated edema in at least one organ selected most drastic when the effect of administering saline from the group consisting of the lung, heart and kidney, alone are taken into account. Clearly, the phallotoxin said organ edema resulting from an interleukin 2 markedly reduces the protein concentration; while the mediated breach of the microvascular barrier of said antamanide greatly decreases and almost neutralizes the organ in a living subject, said method comprising the vascular consequennces of IL-2 administration com 10 step of: pletely. administering an effective amount of an antamanide Overall, therefore, IL-2 (106 units) produces edemain compound to the living subject prior to occurrence the lungs (FIG. 5), heart (FIG. 4), and kidney (FIG. 2) of the interleukin 2-mediated edema, said antama relative to control values. No significant changes over nide compound having the formula controls are found in the spleen (FIG. 1) and liver (FIG. 15 3). Treatment with Phallin B decreases IL-2 induced edema in the lung and heart; however, Phallin B appar ently produces edema in the kidney and the liver. In 20 contrast, treatment with Phallin A prevented IL-2 in duced edema in the lungs, heart, and kidney; moreover, the liver and spleen appear unaffected. The empirical data reported demonstrate that both phalloidin (1 uM) and antamannide (1 uM) can protect 25 against dosage-dependent edemogenic effects of Inter wherein Windividually is an amino acid having at leukin-2 in the heart and in the lung. Antamanide, unlike least one ring structure comprised of not more than phalloidin, always protected against Interleukin-2 medi 6 carbon atoms; ated edemas in at least some organs and tissues; phalloi X individually is an acyclic amino acid comprised of din, however, can improve or worsen IL-2 induced 30 3-9 carbon atoms; edema depending upon the organ examined. These data Y individually is omittable entirely but when present indicate that antamanide and phalloidin individually can is selected from the group consisting of hydrogen, be employed as agent(s) to reduce interleukin-mediated a hydroxyl group, a halogen, and a hydrocarbon edemas produced by iatrogenic therapies. moiety; and The present invention is not to be limited in scope nor 35 Z individually is omittable entirely but when present restricted in form except by the claims appended hereto. is selected from the group consisting of hydrogen, What we claim is: a halogen, and a hydrocarbon moiety. 1. A method for therapeutically treating an interleu 3. A method for therapeutically treating an interleu kin 2-mediated edema in at least one organ selected kin 2-mediated edema in at least one organ selected from the group consisting of the lung, heart and kidney, from the group consisting of the lung, heart and kidney, said organ edema resulting from an interleukin 2 said organ edema resulting from an interleukin 2 mediated breach of the microvascular barrier of said mediated breach of the microvascular barrier of said organ in a living subject, said method comprising the organ in a living subject, said method comprising the step of: step of: administering an effective amount of an antamanide 45 administering an effective amount of an antamanide compound to the living subject after occurrence of compound to the living subject after occurrence of the interleukin 2-mediated edema, said antamanide the interleukin 2-mediated edema, said antamanide compound having the formula compound having the formula 50

wherein W individually is an amino acid having at 60 wherein W individually is an amino acid having a least one ring structure comprised of not more than benzene ring in its structure; 6 carbon atoms; X individually is an amino acid selected from the X individually is an acyclic amino acid comprised of group consisting of valine, alanine, leucine, and 3-9 carbon atoms; isoleucine; Y individually is omittable entirely but when present 65 Y individually is omittable entirely but when present is selected from the group consisting of hydrogen, is selected from the group consisting of hydrogen, a hydroxyl group, a halogen, and a hydrocarbon a hydroxyl group, a halogen, and a hydrocarbon moiety; and moiety; and 5,278,143 15 Z individually is omittable entirely but when present is selected from the group consisting of hydrogen, a halogen, and a hydrocarbon moiety. 4. A method for prophylactically treating an interleu kin 2-mediated edema in at least one organ selected 10 wherein W individually is an amino acid having a from the group consisting of the lung, heart and kidney, benzene ring in its structure; X individually is an amino acid selected from the said organ edema resulting from an interleukin 2 group consisting of valine, alanine, leucine, and 15 isoleucine; Y individually is omittable entirely but when present mediated breach of the microvascular barrier of said is selected from the group consisting of hydrogen, a hydroxyl group, a halogen, and a hydrocarbon organ in a living subject, said method comprising the moiety; and 20 Z individually is omittable entirely but when present step of: is selected from the group consisting of hydrogen, a halogen, and a hydrocarbon moiety. administering an effective amount of an antamanide 5. The method as recited in claim 1, 2, 3, or 4 wherein said administration is parenteral. compound to the living subject prior to occurrence 25 6. The method as recited in claim 1, 2, 3, or 4 wherein said antamanide compound is naturally occurring. of the interleukin 2-mediated edema, said antama 7. The method as recited in claim 1, 2, 3, or 4 wherein said antamanide compound is synthetically generated. nide compound having the formula k s: k 30