WO1989010136A1

WO1989010136A1 - Prophylactic and therapeutic methods for treating edema with phallotoxins

Info

Publication number: WO1989010136A1
Application number: PCT/US1989/001744
Authority: WO
Inventors: David Shepro; J. Steven Alexander
Original assignee: Trustees Of Boston University
Priority date: 1988-04-25
Filing date: 1989-04-25
Publication date: 1989-11-02
Also published as: JPH02501577A; EP0365656A4; AU3539189A; EP0365656A1

Abstract

Unique methods for treating edema in living subjects are provided comprising administering an effective amount of a phallotoxin to the subject. The methods offer prophylactic and therapeutic modes of treatment for both localized and systemic edemas. The phallotoxins may be applied topically or given parenterally. Moreover, a variety of phallotoxins may be employed independently or combined with diverse enhancing agents for treatment of both inflammatory and non-inflammatory edemas.

Description

PROPHYLACTIC AND THERAPEUTIC METHODS FOR TREATING EDEMA WITH PHALLOTOXINS

GOVERNMENTAL SUPPORT Research support for the present invention was provided by U.S. Public Health Service NHLB Grants Numbers HL16714 and HL33104.

FIELD OF THE INVENTION

The present invention is concerned with inflammatory and non-inflammatory associated edemas and is particularly directed to prophylactic and therapeutic methods for treating localized and systemic edemas.

BACKGROUND OF THE INVENTION Edema is the term generally used to describe the accumulation of excess fluid in the intercellular (interstitial) tissue spaces or body cavities. Edema may occur as a localized phenomenon such as the swelling of a leg when the venous outflow is obstructed; or it may be systemic or in congestive heart failure or renal failure. When edema is severe and generalized, there is diffuse swelling of all tissues and organs in the body and particularly pronounced areas are given their own individual names. For example, collection of edema in the peritoneal cavity is known as ascites; accumulations of fluid in the pleural cavity are termed hydrothorax; and edema of the pericardial sac is termed pericardial effusion or hydropericardium. Non-inflammatory edema fluid such as accumulates in heart failure and renal disease is protein poor and referred to as a transudate. In contrast, inflammatory edema related to increased endothelial permeability is protein rich and is caused by the escape of plasma proteins (principally albumin) and polymorphonuclear leukocytes (hereinafter "PMNs") to form an exudate. Edema, whether inflammatory or non-inflammatory in nature, is thus an abnormality in the fluid balance within the microcirculation which includes the small arterioles, capillaries, and post-capillary venules of the circulatory system. Normal fluid balance and exchange is critically dependent on the presence of an intact and metabolically active endothelium. Normal endothelium is a thin, squamous epithelium adapted to permit free, rapid exchange of water and small molecules between plasma and interstitium; but one which limits the passage of plasma proteins with increases in protein size.

The endothelial lining of all arterioles and venules, and most capillaries in the body, is of the continuous type, having an unbroken cytoplasmic layer with closely apposed intercellular junctions. Physiological studies [Renkin, E., Circ. Res. 41:735-743 (1977); Renkin, E., ACTA Physiol. Scand. (Suppl.) 463:81 (1979); Bottaro et a l ., Microvasc. Res. 32:389-398 (1986)] have demonstrated normal endothelial permeability for water and small molecules by the existence of water-filled small pores approximately 6 nanometers (hereinafter "nm") in radius or by slits about 8 nm wide. There is also believed to be a system of larger sized pores about 25 nm in radius which accounts for the small quantities of protein and other large solutes that normally cross the endothelial wall barrier.

A variety of different disturbances can induce a condition of edema. These include: an elevated venous hydrostatic pressure, which may be caused by thrombosis of a vein or any other venous obstruction; hypoproteinemia with reduced plasma oncotic pressure resulting from either inadequate synthesis or increased loss of albumin; increased osmotic pressure of the interstitial fluid due to abnormal accumulation of sodium in the body because renal excretion of sodium cannot keep pace with the intake; failure of the lymphatics to remove fluid and protein adequately from the interstitial space; an increased capillary permeability to fluids and proteins as occurs in the inflammatory response to tissue injury; and an increased mucopolysaccharide content within the interstitial spaces. Currently accepted therapeutic treatments for edema include those biogenic and synthetic pharmacological agents used to treat generalized inflammation, of which edema is a clinical manifestation. Such agents are documented to inhibit the synthesis of pro-inflammatory (pro-phlogistic) metabolites and include such agents as aspirin and ibuprofen (salicylates and propionate derivatives). These agents have a wide scale of effectiveness and, in general, are most valuable in the treatment of minor inflammatory problems that produce only minor, localized edemas. There are few, if any, agents that are therapeutically effective in the treatment of severe, local, and systemic edemas. Furthermore, as far as is known, there is no agent or admixture in use as a prophylactic against these conditions.

It will be noted that until recently the endothelial cells, which constitute the microvasculature, were considered to be functionally passive in nature; in-vivo fluid exchange at the level of the microvasculature was therefore also considered to be functionally passive. Only in 1967 was it proposed that the passage of fluid and solute might occur at interendothelial junctions within the microvasculature, a route also known as the "paracellular" pathway [Karnovsky, M.J., J. Cell Bio. 35:213-236 (1967)]. Subsequent investigations have focused principally upon other aspects of microvascular structural integrity, such as: vesicle transport or transcellular channels which regulate the distribution of integral membrane proteins [Singer and Nicolson, Science 175:720-731 (1972)]; the presence of stress fibers intracellularly which are microfilament bundles composed of actins, myosin, and other contractile proteins [Fujiwara and Pollard, J . Cell. Biol. 71:848 (1976)]; the ability to disrupt endothelial actin cytoskeleton using cytochalasin B with a resulting increase in permeability for plasma proteins [Shasby et al., Circ. Res. 51 : 657-661 (1982)]; the demonstration that serotonin, histamine, and norepinephrine at physiological titers and concentrations inhibit endothelial cell movement [Bottaro et al . , Am. .J. Physiol. 248 : C252-C257 (1985)]; the demonstration that serotonin and norepinephrine stimulate the assembly of stress fibers within endothelial cells whereas histamine produces stress fiber disassembly [Welles et al., J. Cell. Physiol. 123:337-342 (1985) and Inflammation .9:439-450 (1985)]; and, an in-vitro assay which demonstrates that endothelial cells are more effective as a barrier to impede labelled albumin diffusion when compared with cell cultures of vascular smooth muscle or fibroblasts [Bottaro et al., Microvasc. Res.32:389-398 (1986)]. Such investigations have been directed at elucidating the mechanism of action present within the endothelial cytoskeleton; and identifying the role of the cytoskeleton in maintaining microvascular endothelial motility observable as junctional integrity. All these investigations and publications were therefore concerned with only the formulation of a theoretical model for mechanistic cytoskeleton controls and the accumulation of experimental evidence to support the existence of such a mechanistic model.

Remote and completely unrelated to these investigations regarding an active role model for endothelial cells in the microvasculature, were other research efforts directed towards the effects of phalloidin, probably the best known of the toxins isolatable from the poisonous green fungus Amanita phalloides, known as the "green death cap" or deadly agaric" mushroom [Lynen and Wieland, Justus Liebigs Ann. Chem. 533:93-117 (1938); Wieland and Schnabel, Justus Liebigs Ann. Chem. 657 (1962)]. Phalloidin is very rapid in action. High does levels given intramuscularly cause death of mice or rats within one or two hours. The LD₅₀ dose in albino mice is only 3.3 micrograms per gram of body weight given intramuscularly. Phalloidin acts by binding to actin, a cytoskeletal protein [Russo et al., Am. J. Pathol. 109: 133 (1982)]. Reviews of the chemical structure and toxicology of all toxins derivable from Amanita phalloides including phalloidin have been reported in the literature [Wieland and Wieland, Pharmacol. Rev. 11:87-107 (1959); Wieland, T., Fortschr. Chem. Org. Naturst. 25:214-250 (1967); Wieland and Faulstich, Crit. Rev. Biochem. 5:185-260 (1978)].

Sporadically, some investigators have employed phalloidin within in-vitro experiments for its effects upon living cells. For example, the interaction of phalloidin with actin was one of the earliest reported demonstrations [Lengsfeld, A.M., Proc. Natl. Acad. Sci. USA 71:2803-2807 (1974)]. Subsequently, it was shown that phalloidin treatment induces changes in the intercellular junctions of rat hepatocytes [Montsano et al., J. Cell. Biol. 67:310-319 (1975)]. More recently, phalloidin was employed to increase the resistance of Necturus gallbladder epithelium to the passage of an electrical current [Bentzelet al., Amer. J . Physiol. 239:C75-C89 (1980)]. The use of phalloidin in such investigative studies has therefore been primarily as a research tool by which to further characterize and elucidate the mechanism of cytoskeleton action within living cells.

SUMMARY OF THE INVENTION

The present invention provides methods for therapeutically or prophylactically treating edema in a living subject. The method for therapeutically treating edema comprises the step of administering an effective amount of a phallotoxin to the subject after occurrence of the edema. The method for prophylactically treating edema comprises the step of administering an effective amount of a phallotoxin to the subject prior to the occurrence of edema. Either methodology inhibits the permeability of fluid, macromolecules, and blood cells across the microvasculature thereby acting directly on the clinical edema and avoiding indirect metabolic cascades and pathways. The prophylactic methodology can be employed in settings where iatrogenic-induced edema typically occurs such as with the use of clamps and/or tourniquets. The therapeutic methodology can be used to attenuate an inflammatory response as well as a non-inflammatory reaction. Moreover, the phallotoxins may be used alone or in combination with other substances known to affect cellular metabolic pathways as a synergistic mixture in either method. BRIEF DESCRIPTION OF THE FIGURES The present invention may be more easily and completely understood when taken in conjunction with the accompanying drawing, in which: Fig. 1 is a graph illustrating the therapeutic improvement in pulmonary function of living sheep with histamine induced edema after intravenous infusion of phalloidin;

Fig. 2 is a graph illustrating the increase in cell flattening as a function of increased concentration of phalloidin;

Fig. 3 is a graph illustrating the improvement in endothelial barrier function with increasing concentrations of phalloidin; Fig. 4 is a graph illustrating the effect of phalloidin pretreatment against histamine permeability in endothelial cells;

Fig. 5 is a graph illustrating the prophylactic effects of phalloidin pretreatment against bradykinin permeability in endothelial cells;

Fig. 6 is a graph illustrating the prophylactic effects of phalloidin pretreatment against cytochalasin B permeability in endothelial cells; and Fig. 7 is a graph illustrating the prophylactic effects of phalloidin pretreatment against thromboxane A₂ analogues in endothelial cells.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is a general methodology for prophylactically or therapeutically treating localized or systemic edemas in a living subject which comprises the step of administering an effective amount of a phallotoxin to the living subject either before or after occurrence of the edema in the subject. Phallotoxins are a class of compounds isolated from Amanita phalloides and include the bicyclic heptapeptides phalloidin, phallacidin, phalloin, phallisin, prophalloin, phallisacin, and phallacin. The composition and individual methods of preparation for each member within the phallotoxin class of compounds are conventionally known and published in the scientific literature [Wieland et al ., Pharmacol. Rev. 11:87-107 (1959); Wieland et al., Crit. Rev. Biochem. 5:185-260 (1978)]. Each toxin member within the class phallotoxins is individually active and effective in varying degree; and each acts directly on the cytoskeleton of the cell and not via an indirect metabolic cascade for control of edemas. Within the class, phalloidin is presently considered to be the most effective composition; and for this reason, the remainder of the detailed description will focus upon and utilize phalloidin alone as the best representative of the entire class. It will be expressly understood, however, that any of the other members within the class phallotoxins may be similarly employed and used effectively as needed or desired. In-vivo treatment with phalloidin and other phallotoxins is intended to be a general methodology for treatment of edemas in living subjects, particularly humans. The scope of effective treatment using the present invention includes: both prophylactic and therapeutic applications; treatment of localized or systemic edemas; and phallotoxin treatment given independently or in combination with other medical and/or surgical modalities. The present invention is useful and effective with any one or any combination of these parameters. Any of the phallotoxins may be administered to the living subject by one of two different routes: topically by direct application to the skin of the subject; and parenterally by injection or perfusion. If the phallotoxin (independently or in combination with other substances) is to be applied topically, the phallotoxin can be admixed in a pharmacologically inert topical carrier such as a gel, an ointment, a lotion, or a cream; and includes such carriers as water, glycerol, alcohol, propylene glycol, fatty alcohols, triglycerides, fatty acid esters, or mineral oils. Other possible topical carriers include liquid petrolatum, isopropylpalmitate, polyethylene glycol, ethanol, polyoxyethylene monolaurate in water, sodium lauryl sulfate in water, and the like. Other mat erial s such as anti-oxidants, humectants, viscosity stabilizers, and the like may also be added as desired or necessary. In addition, it is expected (and in many instances desirable) that the phallotoxin be disposed within devices places upon, in, or under the skin; such devices include patches and implants which release the active material into the skin or body either by diffusion or by an active release mechanism. Alternatively, if the phallotoxin, independently or combined with other substances, is to be given parenterally, it is expected that the compositions be prepared in sterile form; in multiple or single dose formats; and dispersed in a fluid carrier such as sterile physiological saline or 5% dextrose solutions commonly used with injectables.

In general, the concentration of phallotoxins

(employed independently or combined with other substances) which may be effectively employed for prophylactic and/or therapeutic treatment of living subjects will be: for topical applications, a range concentration from about 1-10 micrograms (hereinafter "ug") per gram of topical carrier. For intravenous, perfusion and other parenteral administration, a concentration range of from about 1.0 micromolar - 0.1 micromolar per L of blood.

Another novel aspect of the present invention is the use of phallotoxins in combination with other substances which function as enhancing agents which increase the activity of the phallotoxins. While many of these enhancing agents are themselves recognized to be either vascularly ineffective or to actually increase the permeability of the microvasculature, when in admixture with phallotoxins, a synergistic admixture is functionally produced which provides an enhanced therapeutic result and treatment of edema which in many instances is superior to the effect of phallotoxin treatment alone. Such enhancing agents include the following: cytochalasins, such as cytochalasin B; free radical scavengers such as superoxide dismutase, mannitol, dimethylthiourea, bis-hydroxy toluene, bishydroxy anisole, vitamins C and E; calcium channel antagonists such as nifedipine, nitrendipine, verapamil, and lanthanum (La ⁺³) ions; and antamanide (a chemical relative of phalloidin).

In addition, when phallotoxins are employed for prophylactic or therapeutic treatment of an edema which is a coincidental characteristic of an inflammatory reaction , it is most desirable that the phallotoxin be administered in admixture with a variety of anti-inflammatory compounds. These include: aspirin, ibuprofen, thromboxane synthase inhibitors; receptor antagonists for the thromboxanes; prostanoid metabolic drugs; steroids; and superoxide and free radical scavengers. A major advantage of the present invention is its ability to prophylactically or therapeutically treat edema, either at localized sites or systemically. To demonstrate the variety of uses and clinical circumstances in which the present methodology can be beneficially employed, a representative, but incomplete listing is provided by Table I below.

Table I

(a) Topical Applications

(1) Treatment of second and third degree burns

(2) Skin cancer such as malignant melanoma and Kaposi's sarcoma

(b) Intravenous Applications

(1) Skin flaps - modify swelling and maintain perfusion (prevention)

(2) Trauma - modify swelling following fractures, crush injuries; prevent critical reduction in blood flow due to "compartment syndrome" in arms and legs (treatment)

(3) Tourniquet use during extremity surgery to minimize reperfusion swelling (prevention)

(4) Revascularization following occlusion of the coronary circulation (by fibrinolytic therapy or coronary artery bypass surgery); renal arterial occlusion; ileofemoral occlusion (prevention)

(5) Organ transplantation - treatment of donor and recipient to minimize swelling of the transplanted organ, thereby preserving function (prevention)

(6) Low flow states associated with hemorrhage and sepsis - to prevent generalized edema; to prevent renal cortical-medulary swelling, reduction of blood flow and acute tubular necrosis (treatment)

(7) Adult respiratory distress syndrome (ARDS) to prevent its occurrence that is permeability pulmonary edema in the following settings

(treatment):

(a) Lung contusion

(b) Acid aspiration

(c) Smoke inhalation (d) Reperfusion of ischemic tissue:

(1) lower torso

(2) gastro-intestinal tract

(e) Septicemia and other severe infections of the chest or abdomen (f) Pancreatitis Table I ( Cont ' d )

(8) Burn edema - to minimize fluid loss into the second or third degree burn (treatment)

(9) Ascites and pleural effusion secondary to inflammation or tumor seeding of the peritoneal or pleural spaces (treatment)

(10) Arthritis - osteo and rheumatoid, to prevent joint swellings and effusions (treatment)

(c) Intraarticular

Treatment of inflammatory edema of osteoarthritis and rheumatoid arthritis (treatemnt)

(d) Intrapleural/Intraperitoneal

Treatment of pleural effusions and ascites secondary to inflammation or tumor nodules

(e) Rectal Enemas to be used to treat ulcerative colitis

(treatment)

(f) Endodontics

Prevention of post-surgical inflammatory permeability such as occurs in root canal procedures (treatment)

To demonstrate the efficacy and value of the novel methods comprising the present invention for prophylactic and/or therapeutic treatment of localized and systemic edemas, a variety of in-vivo and in-vitro experiments will be described. It will be clearly understood, however, that these experimental examples are merely representative of the general conditions, uses, and advantages provided by the present invention; and serve merely to illustrate the variety of operative conditions and diverse applications with which the methods can be usefully employed. Under no circumstances, however, are the specific test conditions or empirically obtained results to be deemed as restricting or limiting the present invention in any manner.

IN-VIVO EXPERIMENTS

Experiment 1: Therapeutic Treatment of Pulmonary Edema

Sheep Preparation: Female animals (n=8) weighing 25 to 38 kg were prepared with a chronic lung lymph fistulae according to a modification of the technique described by Staub et al. [ J . Surg. Res. 19:315 (1975)]. During surgery animals were anesthetized with IV pentobarbitol sodium 15 mg/kg, paralyzed with 2 mg pancuronium bromide, intubated and mechanically ventilated with a Harvard respirator using room air. Through a right posterolateral thoracotomy the efferent duct of the caudal mediastinal lymph node was cannulated with a heparinized silastic catheter (No. 602-155, Dow Corning Corporation, Midland, Michigan). The distal portion of the lymph node, just below the level of the inferior pulmonary ligament was transected and ligated. All visible systemic lymph tributaries to its proximal portion were cauterized or ligated to minimize extra-pulmonary contamination of collected lymph. The thoracotomy was closed and the lymphatic cannula was exteriorized through the chest wall. A thermistor tipped pulmonary arterial (Electro-Cath Corporation, Rathway, New Jersey) and a central venous catheter were introduced through the right internal jugular vein. The aorta was cannulated via the adjacent carotid artery. After a recovery period of 4 to 5 days when the animals appeared vigorous, were afebrile, and had a steady flow of blood free lymph, the experiment was conducted. Cardiopulmonary Function: Strain-gauage transducers (Model D-240, Bently Laboratories, Inc., Irvine, California) were used to measure the following pressures in the sheep: mean arterial (MAP), mean pulmonary arterial, wedge (PAWP). The pulmonary microvascular pressure (Pmv) was calculated from the Gaar equation, Pmv-PAWP + 0.4 (MPAP-PAWP). Pulse rate was determined from an arterial pressure trace. Cardiac output was measured in triplicate by thermodilation (Model 5000, Electro-Cath Corporation, Rahway, New Jersey). Blood gases, pH, oxygen saturation, and hemoglobin of arterial and mixed venous blood were measured with Calak and Severinghaus electrodes and by spectrophotometry using extinction coefficients specific to sheep blood (Model 813 and 282, Instrumentation Laboratory, Lexington, Massachusetts).

Hematology: Circulating platelets and white blood cells (WBC) were counted by means of phase microscopy. Differential counts were made on Wright's stained blood smears. WBC were counted in lymphocyte clearance. Biochemical Assays: Plasma and lymph concentrations of thromboxane B2 and 6-keto-PGF₁α, the stable hydrolysis products of thromboxane A„ (hereinafter "TXA ") and prostacyclin (hereinafter "PGI₂"), were measured in duplicate by radioimmunoassay. Blood was drawn into cooled syringes containing ethylene diamine tetracetic acid (hereinafter "EDTA") and aspirin. The blood was immediately centrifuged at 1,500 x gravity at 4°C for 20 minutes, the plasma separated and stored at -20°C until assayed.

Lung lymph was collected at 30 minute intervals in cold graduated tubes containing EDTA and aspirin. The lymph was then centrifuged at 1,500 x gravity and 4°C for 20 minutes and the supernatant separated and stored at -20°C until assayed for TXB„ and 6-keto-PGF₁α. Lymph (L) and plasma (P) total protein concentrations were determined in duplicate by the spectrophotometric protein dye method described by Bradford. The L/P protein ratio was calculated and multiplied by lymph flow (QL) to obtain the lymph protein clearance.

Experimental Protocol

Initially, each female sheep prepared in the above-identified manner was perfused with histamine at a concentration rate of 2.0 ug/kg min for 1 hour. This created a histamine-induced pulmonary edema in each animal. The experimental animals received an intravenous infusion of phalloidin at a concentration of 3.1 ng/kg min over a period of 6 hours duration. During this infusion period, the experimental animals were awake and permitted free access to food and water. In addition, the animals were allowed to stand or recline as desired and the transducers were leveled as necessary. The biochemical data of control sheep and phalloidin treated sheep evaluated over a 6 hour period is provided by Table II below. The differences in pulmonary function between control sheep and phalloidin sheep over the same 6 hour period are graphically illustrated by Fig. 1. All the data reveal that the histamine-induced pulmonary edema was significantly reversed to normal, baseline levels by the infusion of phalloidin over the entire 6 hour test period. The empirical data thus unequivocally prove and demonstrate that phalloidin and phallotoxins as a class are functional and effective as a therapeutic treatment for edema within living subjects.

in in IN-VITRO EXPERIMENTAL SERIES

Experiment 2: Non-Toxic Effects

Cell Preparation, Culture, and Harvesting: Bovine abdominal aortas were clamped and tied in-situ and dissected free of the supporting adventitia. Aortas were transported from the abattoir on ice. Endothelial cells were harvested by gentle ablation of the endothelium following the technique of Shepro et al. [reference citation]; digested with 0.1% collagenase; and then pelleted and seeded onto 100 mm tissue culture dishes using conventional techniques. The cell medium consisted of Dulbecco's modified Eagle's medium (hereinafter "DME") at pH 7.4 supplemented with 10% fetal calf serum (hereinafter "FCS"), 0.1% of penicillin, 0.7% streptomycin, 0.1% amphotericin, and 0.1% glutamine. The cell media was replaced every 3 days. The cells were incubated at 37°C in a 10% carbon dioxide, 90% ambient atmosphere. The isolation procedure, cobblestone morphology, positive uptake of Dil-acetylated low density lipoprotein and positive staining for the presence of factor VIII related antigen, document and prove that the cells in culture were in fact bovine aortic endothelial cells (hereinafter "BAEC"). Microfilament And Cell Surface Area Measurement:

To demonstrate the effect of phalloidin upon endothelial cell shape, BAEC were seeded onto 1.2 centimeter glass coverslips and allowed to grow to 50% confluency in the conventional manner. This amount of confluency permits excellent visualization of cell spreading and stressed fibers. Individual confluent layers of cells were then treated with either 10^-6, 10^-8, or 10^-10 M phalloidin for 30 minutes duration; then fixed in 3.7% phosphate-buf fered formaldehyde (pH 7.4) for 15 minutes. The cells on each coverslip were then permeabilized in extraction buffer (composed of 0.5 M KCl, 1% triton X-100, 10 mM MgCl₂, and 1 mg/ml tosyl-arginine methyl ester [hereinafter "TAME"]); 17 ug/ml toluene-sulfonyl fluoride (hereinafter "TSF"); and 0.25 mg/ml DNAse 1 in PBS for a period of 10 minutes. Subsequently, this extraction buffer was removed and the cells washed three times in sequence with PBS. The cells were then stained with Rhodamine-phalloidin (an F-active specific fluorescent probe, Molecular Probes Inc., Junction City, Oregon) at 1 unit/200 ul per coverslip for 30 minutes duration. Subsequently, the coverslips were washed five times consecutively in PBS; mounted in a 1:1 ratio mixture of PBS/glycerol; and sealed. The cultures cells on each coverslip were illuminated for fluorescence microscopy. The results showed that endothelial cells treated with phalloidin demonstrated an increased surface area and cell perimeter, the roundness of a cell being expressed as a ratio of cell perimeter to area. When BAEC are treated with phalloidin, the ratio of cell perimeter to cell area diminishes with increasing concentrations of phalloidin as is graphically illustrated by Fig. 2. In each instance, the treated cells have undergone substantial flattening and border extension.

In addition, the use of phalloidin at concentrations of 10^-6, 10^-8, and 10^-10 M respectively were found to be non-toxic to the cultured cells. This absence of toxicity was measured and demonstrated by ethidium bromide-fluorescein acetate testing following the method of Edidim [J. Immunol. 104:1303-1306 (1970)]. The empirical results demonstrate that phallotoxin used at concentrations b etween 10 and 10 M are no t t oxi c to end o thel ial cell s .

Experiment 3: Improvement of Endothelial Barrier Function Microcarrier Bead Culture: Bovine aortic endothelial cells (BAEC) were prepared, cultured, and harvested as described within Experiment 2. Subsequently, BAEC were subcultured a maximum of three times before seeding onto Cytodex 3 microcarrier beads (Pharmacia, Inc.). Microcarrier beads were suspended in cell media at a concentration of 40,000 microcarriers per milliliter. Beads were suspended by stirring, using a magnetic stirrer. BAEC were seeded onto the beads at the minimum density of 15 cells per bead and allowed to reach confluency. Subsequently, BAEC covered beads were allowed to incubate for 6 days postconfluency to obtain near maximum barrier function of the cell monolayers on the beads. After this incubation period, the BAEC covered beads were ready for use within permeability experiments. The ability of BAEC to form a functional barrier to the exchange of different marker substances between the bead interior and the surrounding media was tested in accordance with the methods of Boiadjeva et al. [Lab. Invest. 50:239-246 (1984)] as modified by Bottaro et al. [Microvasc. Res. 32:389-398 (1986)].

Preparation of Permeability Assay Media: The identifying marker for permeability transfer between the bead interior of BAEC-covered beads and the surrounding culture medium was a conjugate of trypan blue dye and bovine serum albumin (hereinafter "TBA"). The molecular weight of this TBA identifying marker has been reported in the literature as being 100,000 daltons and has been demonstrated to be a useful identification marker for paracellular exchange. The TBA solution was prepared as a 2 x concentrate in PBS (pH 7.4) and stored at 4 C until required for use. The final concentration of TBA in the surrounding cell culture medium was 0.2% trypan blue and 0.4% albumin in all instances.

Permeability Assay Procedure: BAEC barrier function was measured by TBA uptake per bead; therefore it is necessary to standardize the number of beads per milliliter. Cytodex 3 beads are composed of cross-linked dextrans and were standardized using an assay for total carbohydrate content of a 50 microliter (hereinafter "ml") aliquot of bead suspension using the Kochert method [The Handbook of Phycological Methods (J.A. Hellebust and J.S. Craigie, editors) Cambridge University Press, Cambridge, 1978, pages 95-97]. In brief, the standardization utilized 50 ul bead samples which were dissolved in 5 ml of concentrated sulfuric acid for 15 minutes and then combined with 2 ml of 2.25% phenol. This produced a colored reaction product which absorbed light at 485 nm. Standard curves for absorbance as a function of bead concentration provides a linear relationship as a basis for comparison and extrapolation. Test samples were then compared with the standard curve to assess bead number per vial or cuvette. Controls for each test sample consisted of untreated BAEC-covered beads to assess the maximum possible uptake of TBA in the absence of other treatments.

BAEC barrier function was then assessed in the following manner: 3.0 ml of BAEC-covered microcarrier beads at a density of 40,000 beads per milliliter were aliquotted into a 4.5 ml test vial in duplicate. The TBA and the substante under test were then added to the culture medium. Alternately, beads could be pretreated with the substance under test prior to the addition of TBA. Vials containing the BAEC-covered beads were agitated and maintained under proper culture conditions. At preselected time intervals, triplicate 150 ul aliquots of the BAEC-covered beads were then removed from each vial and placed on a cushion containing a 3:1 ratio mixture of dibutyl/dioctyl pthalate. The cell suspension was then centrifuged for 30 seconds at 15,000 x gravity. Centrifugation effectively terminates the TBA uptake by the beads by separating the dye mixture from the beads. The dye concentration in the supernatant after centrifugation was assayed by mixing 100 ul of the supernatant with 900 ul of distilled water and measuring the absorbance of the fluid at 595 nm .

This assay procedure uses colorimetry to measure the TBA dye absorbance by the beads. If a test substance reduces BAEC barrier function, the beads would take up more dye in comparison to the controls; and the supernatant would then contain less dye in comparison with controls. Conversely, if a test substance increases barrier function, this would cause a reduced uptake of dye by the beads, and the dye concentration of the supernatant would be greater than that of the controls.

Increases In Barrier Function By Phalloidin:

The prepared aliquots of BAEC-covered microcarrier beads were individually combined with 10 ^-6, 10^-8, and 10^-10 M of phalloidin. The experimental controls received no phalloidin treatment whatsoever. TBA was added to each test aliquot and the permeability assay conducted in the manner described above. Centrifugation and test of supernatants was performed at 30 minutes following the initial addition of phal loidin to each test aliquot of BAEC-covered beads.

The results are graphically illustrated by Fig. 3.

Clearly, the addition of phalloidin in concentrations from 10^-6-10^-10 M improves and enhances resistance to permeability in endothelial cells and markedly increases cell barrier function in comparison to untreated controls.

Experiment 4: Protection Against Cellular Effects Of Histamine And Bradykinin Endothelial barrier function was evaluated using the permeability assay protocol previously described within Experiment 3 above. Aliquots of BAEC-covered beads serving as test samples were initially pretreated for 30 minutes with 10^-8 M phalloidin. Control aliquots received no phalloidin pretreatment whatsoever. Subsequently, test aliquots and control aliquots were combined with 10^-6 M of histamine, an agent known to increase cell permeability and to reduce cell barrier function. Test samples and controls were then incubated and cultured as previously described above. Subsequently, all aliquots were evaluated for cell barrier function using the method described previously. The results are graphically illustrated by Fig. 4 which unequivocally demonstrates that phalloidin pretreatment protects against histamine permeability effects.

A second experimental series was then performed substituting bradykinin in place of histamine as the substance known to increase cell permeability and to reduce barrier function. As before, some test samples were pretreated with 10^-8 M of phalloidin for 30 minutes while other test aliquots of

BAEC-covered beads received no phalloidin pretreatment whatsoever. Subsequently, bradykinin was added to all test aliquots at a concentration of 10^-6 M. The controls did not receive any bradykinin or phalloidin at any time. The results are graphically illustrated by Fig. 5. Again, it is clearly demonstrated that pretreatment with phalloidin protects against bradykinin permeability and avoids reduction of cell barrier function.

Experiment 5: Protection Against Cytochalasin B Permeability This experimental series again employed

BAEC-covered beads and the permeability assay protocol previously described within Experiment 3. Initially, aliquots of BAEC-covered beads in culture were pretreated with 10^-8 of phalloidin as previously described. Other test aliquots of BAEC-covered beads received no phalloidin pretreatment whatsoever. Subsequently, all aliquots of test cells were combined with 10 M of cytochalasin B, a known microfilament disrupting agent able to substantially reduce cell barrier function of endothelial cells. Subsequently, all test samples and controls were evaluated using the permeability assay protocol described above. The results are graphically illustrated by Fig. 6. Clearly, pretreatment with phalloidin protects against cytochalasin B permeability.

Another experimental series was performed using aliquots of BAEC-covered beads, cytochalasin B, and phalloidin. This series of experiments, however, employed a mixture of 10^-6 M cytochalasin and 10^-8 M palloidin in combination as a test reactant and compared the effects of this mixture against the increased permeability effects of cytochalasin B alone. Moreover, in this experimental series, there was no pretreatment with phalloidin whatsoever. Accordingly, aliquots of BAEC-covered beads were combined with the reactant mixture (phalloidin and cytochalasin B) and with cytochalasin B as individual reaction mixtures. The empirical data demonstrates in significant (P 0.05) increases in endothelial barrier function over cytochalasin B alone.

Experiment 6: Protection Against Thromboxane A₂ Permeability

BAEC-covered beads were prepared and tested by permeability assay as described previously within Experiment 3. Within this experimental series , some test aliquots of BAEC-covered beads were initially pretreated for 30 minutes with phalloidin at a concentration of 10^-8M. Other test aliquots of

BAEC-covered beads received no phalloidin pretreatment whatsoever. Subsequently, each of these test aliquots received 10^-6 M of a thromboxane A₂ analog,

9,11-dideoxy-epoxymethano-PGF1_a (hereinafter "TXA₂ analog"). All aliquots of cell covered beads were then allowed to incubate as previously described above. Subsequently, each test sample and control was evaluated by the permeability assay protocol previously described. The empirical results are graphically illustrated by Fig. 7. Clearly, pretreatment with phalloidin resulted in protection against the increased permeability effects of TXA₂ analog; in comparison, the effect of TXA₂ analog without phalloidin pretreatment, resulted in significant (P 0.05) increases in BAEC permeability compared with untreated controls. The totality of the empirical data presented herein, whether in-vivo or in-vitro, is deemed to demonstrate and prove the effectiveness and function of the present methodology as a whole. The empirical data provides clear and unequivocal evidence for: the utility of the present methods within living subjects; the capabilities and advantages of the present invention as both prophylactic and therapeutic treatment methods; and the effectiveness of the present methodology within localized or systemic use circumstances.

The present invention is not to be restricted in form nor limited in scope except by the claims appended hereto.

Claims

What we claim is:

1. A method for therapeutically treating edema in a living subject, said method comprising the step of: administering an effective amount of a phallotoxin to the subject after occurrence of the edema.

2. A method for prophylactically treating edema in a living subject, said method comprising the step of: administering an effective amount of a phallotoxin to the living subject prior to the occurrence of the edema.

3. The method as recited in claim 1 or 2 wherein said edema is localized.

4. The method as recited in claim 1 or 2 wherein said edema is systemic.

5. The method as recited in claim 1 or 2 wherein said edema is clinically recognized as pulmonary edema.

6. The method as recited in claim 1 or 2 wherein said phallotoxin is selected from the group consisting of phalloidin, phallacidin, phalloin, phallisin, prophalloin, phallisacin, and phallacin.

7. The method as recited in claim 1 or 2 wherein said administration is topical.

8. The method as recited in claim 1 or 2 wherein said administration is parenteral.

9. The method as recited in claim 8 wherein said administration is selected from the group consisting of intravenous, intramuscular, and intraperitoneal administration.

10. The method as recited in claim 1 or 2 wherein said phallotoxin is combined with an enhancing agent.

11. The method as recited in claim 10 wherein said enhancing agent is selected from the group consisting of cytochalasins, free radical scavengers, calcium channel antagonists, antamanides, and anti-inflammatory compounds.