WO1994012210A1

WO1994012210A1 - Anti-edlf antibody, composition thereof and method of diagnosing and treating cardiac arrhythmias, hypertension

Info

Publication number: WO1994012210A1
Application number: PCT/US1993/011365
Authority: WO
Inventors: Alexei Y. Bagrov
Original assignee: Bagrov Alexei Y
Priority date: 1992-12-02
Filing date: 1993-11-29
Publication date: 1994-06-09
Also published as: AU5728494A

Abstract

New compound and antibody compositions used in preventing and treating hypertension and cardiac arrhythmias, method of preparation and method of diagnosis, and/or predicting onset of cardiac arrhythmias brought about by various pathological conditions. The invention is further directed to antibodies prepared from steroid compounds derived from the venom and tissues of the Bufo marinus toad for blocking endogenous digoxin-like factors found in the plasma of mammal.

Description

ANTI-EDLF ANTIBODY, COMPOSITION THEREOF AND METHOD OF DIAGNOSING AND TREATING CARDIAC ARRHYTHMIAS,

HYPERTENSION BRIEF DESCRIPTION OF THE INVENTION

The invention is directed to new compounds and antibody compositions effective in preventing and treating hypertension and cardiac arrhythmias, method of preparation and method of diagnosis, and/or

predicting onset of cardiac arrhythmia brought

about by various pathological conditions. The

invention is further directed to antibodies prepared from steroid compounds derived from the venom and tissues of the Bufo marinus toad for blocking

endogenous digoxin-like factors found in the plasma of mammal.

BACKGROUND OF THE INVENTION

Hypertension is the primary risk factor for coronary, cerebral and renal vascular diseases which cause over half of all deaths in the United States. It has been estimated that the number of hypertensive patients in the United States alone is substantially 57.7 million and on the rise. The widespread

awareness of the danger of elevated blood pressure has become the most frequent reason for visits to

physicians. No single or specific cause is known for the hypertension referred to as primary (essential) hypertension. Primary hypertension has been attributed to such causes as hemo dynamic pattern, genetic

predisposition, vascular hypertrophy, hyperinsulinemia, defects in cell transport or binding, defects in the renin-angiotensin system (low-renin or high renin hypertension) and along with insulin, angiotensin and natriuretic hormone, catecholamines arising in response to stress are known to be pressor-growth promotors.

Increased sympathetic nervous activity may raise the blood pressure in a number of ways, for example, either alone or in concert with stimulation of renin release by catecholamines, causing arteriolar and venous

constriction, by increasing cardiac output, or by altering the normal renal pressure-volume relationship.

Primary hypertension is also associated with, for example, obesity, sleep apnea, physical inactivity, alcohol intake, smoking, diabetes mellitus, polycythemia and gout. Secondary forms of hypertension may arise from oral contraceptive use and parenchymal renal disease; renovascular hypertension caused by, for example, atheroschlerotic disease, tumors

(renin-secretory tumors); Cushing Syndrome; heart surgery; and pregnancy. Chronic hypertension and renal disease during pregnancy may progress into eclampsia, a primary cause of fetal death.

It has been theorized that blood serum and various mammalian tissues contain a substance,

biologically and immunoreactively, similar to digitalis glycosides and digoxin (ouabain)-like which have been labeled endogenous digoxin-like factors (EDLF). This theory has been supported in recent years by

considerable evidence of a causal role for sodium in the genesis of hypertension. The evidence includes the finding of increased intracellular sodium in hypertensive mammals. Increases have also been noted in normotensive children of hypertensive parents. It has been discovered that an increased fluid volume stimulates the secretion of EDLF that inhibits the Na⁺ ,K⁺ -ATPase pump. The inhibition is brought about by the reaction of the EDLF with the alpha-subunit of the ouabain-sensitive-magnesium-dependant,

Na⁺ ,K⁺ -ATPase in a manner similar to the digitalis glycosides. In the case of renovascular types of hypertension, inhibition of the sodium pump increases renal sodium excretion and restores vascular volume while at the same time leading to hypertension by increasing intracellular sodium content by potentiating preexisting vasoconstriction and finally initiating a new circle in the pathogenesis of hypertension.

Increased plasma concentrations of EDLF have been discovered in hypertension caused by other physical and pathological conditions. Consequently, it was

discovered that the administration of an antidigoxin anti serum to hypertensive animals causes a pronounced decrease in the blood pressure.

The exact chemical nature, as well as the site of origin, of EDLF is not known. It has been proposed that endogenous digoxin is a peptide originating in the hypothalamus. It has also been reported that EDLF originates in the adrenals and the heart. It has been shown that the EDLF substance exists in several

different molecular forms, at least one of the forms being steroidal in nature. Confirming this, it was discovered that several steroids with digitalis-like immunoreactivity and ability to inhibit Na⁺,K⁺-ATPase were identified in various amphibia tissues.

It was discovered that EDLF has direct effects on the heart and that the mammalian heart contains a substance with digoxin-like immunoreactivity and other properties of digitalis. The existence of the

different subpopulation of the high-affinity receptors for digitalis in myocardium and neural endings in the heart indicated the existence of an endogenous ligand(s) at these receptor sites. It is known that an overdose of digitalis glycosides provokes cardiac arrhythmias, including ventricular tachycardia and ventricular fibrillation, rather than increasing cardiac

contractility. Acute myocardial ischemia (AMI)

sensitizes the myocardium to the arrhythmogenie effect of digitalis and is associated with both the inhibition of myocardial sodium pump activity and with the loss of digitalis specific receptors. Based upon this prior knowledge, it was hypothesized that the increased plasma concentrations of EDLF contributed to the origin of the hypersensitivity of the ischemic myocardium to digitalis and that EDLF participates in the genesis of myocardial ischemia-induced arrhythmias. Based upon this

hypothesis, it was discovered that the plasma

concentrations of digoxin-like immunoreactivity, (for example) EDLF, was significantly increased after a first transmural myocardial infarction; it was further

discovered that acute myocardial ischemia is associated with a marked increase in the concentration of EDLF which increase occurs in parallel with the onset of ventricular arrhythmias.

DETAILED DESCRIPTION OF THE INVENTION

It was unexpectedly discovered that the antiEDLF antibody prepared from marinobufagin, in accordance with the increased plasma concentration of EDLF, for example arrhythmias, and prevented hypertension. The invention enabled the method of diagnosis, and or predicting the onset of cardiac arrhythmias by various pathological conditions. It was formerly discovered that antibodies prepared from the steroid compounds derived from

marinobufagin effectively blocked endogenous digoxin-like factors found in the plasma of man.

Treatment to prevent or alleviate cardiac

arrhythmias utilizing the antibody of the invention may be by any of the conventional routes of administration, for example, oral, intramuscular, intravenous or rectally. In the preferred embodiment, the antibody is preferably administered in combination with a

pharmaceutically-acceptable carrier which may be solid or liquid, dependant upon choice and route of

administration. Examples of acceptable carriers include, but are not limited to, for example,

physiological saline solution.

In the preferred embodiment, the inventive compounds are administered intravenously. The actual dosage unit will be determined by such generally recognized factors as body weight by patient and, in particular, the severity of the arrhythmia and type of pathological condition the patient might be suffering with. With these considerations in mind, the dosage of a particular patient can be readily determined by the medical practitioner in accordance with the

techniques known in the medical arts.

EXAMPLE I

The polyclonal monospecific antidigoxin antibody of the invention was obtained by immuiizing chincilla rabbits with a digoxin-bovine albumin conjugate. Each animal was injected with 0.5 mg of the conjugate

dissolved in 0.5 ml water and mixed in a ratio of 1:1 with Freund's adjuvant. The mixture was administered by subcutaneous injection in five different locations on the backs of the rabbits over a four-week period.

Antidigoxin immunoglobulin was separated by

immunoaffinity chromatography using CNBr-Cepharosa coupled with digoxin-RNAase conjugate.

EXAMPLE II

Acute myocardial ischemia was set up in

seventy-three adult male Wistar rats anesthetized with sodium pentobarbital (75 mg/kg intramuscul lary) and artifically ventilated via tracheostoma. After

thoracotomy, the left coronary arteries were ligated 1-2 mm distal to their origins. The hearts were

monitored by three standard ECG leads. Test drugs were adminstered into the femoral veins via polyethylene catheters. After fifteen minutes of acute myocardial ischemia, the animals were sacrified by exsanguination. It will be understood by those skilled in the art that the fifteen minute period corresponds to an approximate three to four hour period of myocardial infarction in humans. Blood samples were collected from the abdominal aortas into cooled polyethylene tubes containing 0.1M EDTA and 10 μM phenylmethylsulfonylfluoride (50 μ1 per 4 ml blood). The resulting solution was frozen at -20°C for determination of digoxin-like immunoreactivity

(EDLF) in the plasma. The digoxin-like

immunoreactivity was measured using dissociation

enhanced lanthanide fluoroimmuniassay (DELFIA) kits by LKB, Finland. This assay of digoxin is a solid phase immunoassay, i.e., digoxin is immobilized in the wells of the plate, based on a competition between immobilized digoxin and sample digoxin (in the present case EDLF) for europium-labeled polyclonal antidigoxin antibodies derived from rabbits. Standard and sample (or EDLF) reduce the binding of the europium labeled antibodies to the immobilized digoxin molecules. Finally,

fluorescence in the microtitration strip wells is measured in a resolved LKB-Wallac fluorometer.

Arrhythmia incidence was defined as the total duration of ventricular tachycardia (VT) and ventricular fibrillation (VF) during the fifteen minute postligation period. The animals were divided into five groups as follows:

Group 1. Twelve (12) control, rats subjected only to thoracotomy;

Group 2. Twenty-eight (28) rats pretreated with am intravenous injection 0.2 ml isotonic saline prior to period of acute myocardial ischemia;

Group 3. Fifteen (15) rats pretreated by intravenous injection of 260 mg/kg antidigoxin

immunoglobulin;

Group 4. Five (5) rats pretreated by intravenous injection with 5 mg/kg DIGIBIND (Fab fragments of bovine antidigoxin antibody, a drug produced for the treatment of digoxin overdose by Burroughs Wellcome Co.); and

Group 5. Seven (7) rats pretreated with 40 mg/kg

DIGIBIND.

All pretreatment of the animals was carried out thirty minutes prior to coronary ligation. The control animals were pretreated thirty minutes prior to

thoracotomy.

No heart rhythm disturbances were observed in the control animals. The plasma concentration of

digoxin-like immunoreactivity (EDLF) in the control animals was 0.48±0.09 mg/ml. Acute coronary ligation in Group 2 animals resulted in typical ischemic changes of the ECG, i.e., increase in the R wave, elevation of the ST-T segment and in the onset of ventricular arrhythmias. Average duration of VT and VF in Group 2 was 201±34 sec. Plasma concentration of EDLF ranged

1.13±0.32, p<0.05.

The. Group 3 animals exhibited a reduced average duration of VT and VF to 46+/-18 sec . ( p<0.02 ) and a decr ease in the plasma concentrati on of EDLF , i . e . ,

0.20±0. 06 , p<0 . 05 . The Group 4 animals did not exhibit any change in the incidences of post

ligation arrhythmias.

The average duration of VT and VF was reduced to 74+34 sec. in the Group 5 animals. This difference was statistically nonsignificant when compared with the Group 1 animals with myocardial ischemia.

From this experiment, it was concluded:

1. Acute myocardial ischemia in rats is

associated with a marked increase of the concentration of digoxin-like immunoreactivity.

2. The increase in digoxin-like immunoreactivity occurs in parallel with the onset of ventricular arrhythmias.

3. Pretreatment of the animals with polyclonal antidigoxin rabbit IgG significantly reduces the incidence of arrhythmias and prevents the increase of plasma EDLF associated with coronary ligation. That means that antidigoxin IgG binds the circulating EDLF and prevents the development of its physiological effects.

4. Digibind, even at extremely high

concentrations, was almost inactive in suppressing the ischemia-induced arrhythmias. In our previous

experiments, we compared the effects of our antidigoxin IgG and digibind on the inhibition of the human intact red blood cells Na,K-ATPase by digoxin in these experiments (n+6, detailed description of the method below) digibind was more active than our IgG in

protecting the red blood cell Na,K-ATPase from the inhibition by digoxin. Digibind has a very high

affinity to digoxin (10^-9-10^-10 _m/1). Consequently, having very high affinity to digoxin, digibind is less active against EDLF. That observation suggested to us to produce antibody raised against not digoxin itself but

EDLF, which have shown extraordinary antiarrhythmic effects (Group G).

EXAMPLE III

The Bufo marinus toad poison used in the examples was obtained from venom obtained from the parotid glands of Bufo marinus male and female adult toads obtained from the St. Petersburg, Russia and Riga, Latvia

Zoological Gardens. The venom was extracted by soft pressing on the skin around the glands. The venom crystallizes at room temperature within 24 hours at 23 °C. 800 mg of the crystallized poison was subjected to soaking extraction using 50% ethanol at a temperature of 30°C with periodic shaking over a two-week period. Following the alcoholic extraction, the mixture was filtered through Shott Nr 4 filters. The filtrand was divided into two portions. Each portion was washed with 3ml 50% ethanol. After removal of the filtrate the residue was further extracted with a 1:1 solution of 50% ethanol and chloroform followed by centrifugation in order to obtain chloroform and ethanol phases. The chloroform phases were isolated and extracted,

centrifugation steps were repeated two times after which the chloroform phases were mixed and distilled under vacuum. A dark brown oily residue resulted which was dissolved in 1 ml of ethylacetate. The non-soluble residue was separated by filtration.

A mixture of steroid compounds was obtained and separated by thin-layer chromatography (Silufol VV 254, Sigma Chemicals), plates were preexposed to 1 hour preincubation at 100°C. Ethyl acetate was used as the eluent. Identification of the individual

steroids was performed in UV. A spot corresponding to marinobufin was scraped, divided into three (3) portions and extracted with ethyl acetate. In

parallel series all eleven (11) spots corresponded to the steroids resibufagenin, substance L, bufalin, marinobufagin, telocinobufagin, argentinogenin,

gel lerbrigenin, jamaicogenin, gellerbrigenol,

gamabufitalin, and substance D.

The steroid compounds and substances were

scraped from Silufol plates and treated to the

same procedure as with the marinobufagin. The steroids developed and used in the experiments herein are as follows: Resibufagen % of LD_SO

(mice)

3_β-hydroxy 14,15 poison weight epoxybufadienolide

0.07 inactive

Marinobufagin 10 0.152

3_β 5_β dihydroxy 14,15

epoxybufadienolide

Cinobufagin

3_β hydroxy 16_β acetoxy- -CH₃

14,15 epoxybufadienolide 0.209

Buf alin

3_β 14_β dihydroxy- dienolide 0.7 0. 137

Bufal in

3_β 14_β dihydroxy- dienolide 0.7 0.137

Telocinobufagin

3_β5_β14_β trihydroxybufadienolide

0.102

Gamabufotalin

3_β 11 14_β- trihydroxybufadienolide 0.10

Hel lebrigenin

3_β 5_β 14_β- trihydroxy- 19nor-19 aldehyde0.077 bufadienolide

EXAMPLE IV

The bufo steroids prepared in accordance with Example III are conjugated with larger macromolecules in order to obtain greater immunogenicity for the steroids to produce the antibodies of the invention. 14 mg of a copolymer of vinylpyrolidon, maleic anhydride and maleinic acid (51:48:4, mw 1000) and 2mg of

marinobufagin were dissolved in 1ml anhydrous pyridine and maintained at 30°C for a period of 72 hours. As a result of the reaction, a complex ether bond is

established between the copolymer and marinobufagin, wherein an anhydric group of the copolymer serves as a carbonyl component and the hydroxyl C₃ group of the steroid serves as an alcohol component. Following the reaction the conjugated compound is treated to

thin-layer chromatography (as in Example I).

Disappearances of the marinobufagin zone indicates that the reaction is complete. Pyridine is removed by vacuum filtration. The residue is suspended in 5 ml of

anhydrous benzene filtered and washed with 1 ml of ethanol to remove non-conjugated steroids. The compound is dried under vacuum.

EXAMPLE V

Conjugated compounds of the mixture of steroids of Examp le III were prepared in accordance with the method of Example IV.

EXAMPLE VI

In order to prepare the antibody of the invention, adult Chinchilla rabbits were immunized with a conjugate of a bufo steroids or a conjugate, a mixture of the bufo steroids prepared in accordance with the method of

Examples III and IV. Each animal was subcutaneously injected with 1 mg of the conjugate over a period of five weeks using the procedure described in Example I. After five weeks, venous blood was collected from the ear veins in 20-30 ml increments, twice weekly. The serum was separated and tested for ability to antagonize the in vitro vasoconstrictor effect of marinobufagin and bufo marina venom in isolated rat aortic rings in the manner of the procedure carried out in Example I. It was found that the antiserum significantly decreased the constrictor response of the isolated aortic rings to both marinobufagin and venom.

The inventive immunoglobulins were separated from the whole serum in the following steps:

Step 1. The serum is diluted (1:4) with an acetate buffer (60 mM CH₃COONa-CH₃COOH, pH4). The pH of the solution was adjusted to pH 4.5 using 0.1N NaOH.

Step 2. 25 NL Caprylic (octanoric) acid was slowly added with stirring to 1 ml of the serum solution. The final solution was stirred for thirty (30) minutes followed by centrifuging to separate the proteins of non-immunoglobulins nature.

Step 3. The supernatant from Step 2 was filtered and the filtrate was dissolved, 9:1, in a phosphate buffer solution (150mM NaCl, 3mM KC1, 8mM Na₂HPO₄, 1.5mM KH_zPO₄ , pH 7.2). The pH was adjusted to 7.4 with IN NaOH. The resulting solution was cooled to 4°C followed by the addition of (NH₄)₂SO₄. The

resulting mixture was centrifuged and a precipitate of proteins separated.

Step 4. The precipitate from Step 3 was

dissolved in the minimal amount of distilled water and dialyzed in sevaporl dialysis bags (threshold, protein with m.w.17,000D) against two changes of 1L of

distilled water. Dialysis was controlled by

concentrated BaCl₂ (in the presence of the 50₄ ions we saw undissolvable BaCl₂). The dialysized anti-EDLF immunoglobulin of the invention obtained hereby was used in the tests.

EXAMPLE VII

The polyclonal mono specific an ti marinobufagin antibody of the invention used in the testing of the invention was obtained by immunizing six (6) Chinchilla rabbits with a marinobuf agin-glycoside-protein (BSA) conjugates. Each animal was injected with 0.5 mg of the conjugate dissolved in 0.5 ml water and mixed in a ratio of 1:1 with Freund's adjuvant. The mixture was administered by subcutaneous injection in five different locations on the backs of the rabbits over a four-week period. The inventive immunoglobulins were separated from the whole serum as described in Example VI.

EXAMPLE VIII

The antimarinobufagin antibody of the invention as prepared in accordance with the method of Example V and was tested in accordance with the procedure

described in Example II. Antimarinobufagin antibody blocked the positive, inotropic and arrhythmic effect of the mixture of steroids in isolated, spontaneously constricting rat aorta, and prevented vasoconstrictor action of the mixture of steroids in isolated rat aorta. In fact, the antimarinobufagin antibody presented vasoconstrictor action of the mixture of steroids two effects, namely, anti-arrhythmic and vasoconstriction.

EXAMPLE IX EDLF content in blood serum and tissue was assayed using half area enzyme immunoassay plates coated with BSA (bovine serum albumin) marinobufagin by adding to each well of 50-100 μ1 of lng/ml BSA-EDLF in a buffer (50mM sodium carbonate, ph 8.6). The plates were stored at 4°C for 1-2 days. Unbound BSA-EDLF

conjugate was washed out by washing each well repetitively with rinse solution (0.09/NaCl) containing 0.05%

Tween 20).

The titer of anti-EDLF antibody from immunized rabbits or produced by hybridoma technic was determined on plates as described above. Doubling dilutions of antibody were added to the wells starting from 1:1,000.

The plates were incubated with shaking for 60 minutes at a temperature of 30°C; this allows the anti-EDLF

antibody to bind to BSA-EDLF conjugate attached to a plate. After incubation, the antibody was washed out with four rinses. Antibody which bounded to EDLF remained on the wells being attached.

The 1:1,000 diultions of goat anti-rabbit IgG horseradish peroxidase conjugate were added to each well for 60 minutes with continuous shaking. There- after unbound goat anti-rabbit IgG-peroxidase conjugate is washed away. Then TMB reagent added (50 μ.1 ) to each well. After 15 minutes reaction is stopped log addition of 50 μ1 of 1M H₃ PO₄. Absorbance of each well is measured at 450nm. Standard EDLF ( marinobufagin) curve is plotted (1.10^-9 to 1.10^-5 M/D. Addition of EDLF prevents binding of anti-EDLF antibody to BSA-EDLF conjugate in the well. Consequently, less goat anti- rabbit IgG binds to the well and we see less absorbance at 450 nm.

It will be understood by those skilled in the art that other methods of immunoassay are readily available in the art, for example, radioimmunoassay.

EXAMPLE X

Fifty-four (54) patients who had never taken digitalis drugs and had no known history associated with increased concentrations of EDLF, e.g., severe

hypertension, renal and hepatic diseases, and endocrine dysfunctions, who were admitted to the coronary care unit of the Djanelidze First Aid Institute with a first time transmural acute myocardial ischemia (AMI) were studied. Also not included in the study were patients who received systemic thrombolytic therapy. The

diagnosis of AMI was based upon a typical chest pain of at least thirty minutes duration, ST segment elevation on the ECG with subsequent development of Q waves in the involved leads (Minnesota Codes 1-1-1, 1-2-5, 1-2-6, and 1-2-7), at a two-fold increase of plasma total creative phosphokinase and lactate dehydrogenase.

Patients known to have unstable angina pectoris and suspected AMI, together with healthy donors, served as control groups. Venous blood samples were obtained from the patients each day for ten days and on the fourteenth day following the diagnosis of AMI. Blood was collected in cooled polyethylene tubes containing 10mm

phenyl methyl sulfonyl fluoride in 0.1 μmol liter-1 EDTA and plasma. The mixtu was frozen at -18°C prior to assay. Plasma concentrations of EDLF were measured using the dissociation-enhanced lanthanide

fluoroimmuniassay method digoxin kit and expressed as ng/ml of digoxin equivalents. The results were analyzed statistically using Student's t-test.

Fifty-four Caucasian patients (47 male, 7 females) ages 39 to 72 years, (mean age 54.5 years) with AMI, 16 Caucasian male patients with unstable angina pectoris and suspected AMI ranging in age from 40 to 67 years

(mean age 50.4 years), and eight healthy donors (3 males, 5 females), mean age 39.3 years, were enrolled in the study.

Plasma concentrations of digoxin-like

immunoreactivity in patients during the first 24 hours following onset of AMI were significantly increased (1.25± 0.26 ng/ml) as compared with the healthy

controls (0.34± 0.08 ng/ml) and patients with unstable angina pectoris (0.04± 0.06 ng/ml). The condition of seven of the patients within the first 24 hours after onset of AMI was complicated by primary ventricular fibrillation. In these patients, the concentrations of EDLF was significantly higher (2.54± 0.67 ng/ml) than in the 47 patients with AMI who did not experience ventricular fibrillation (1.05± 0.27 ng/ml), ρ<0.05). During the first 24-hour period of time, 14 of the patients exhibited manifestations of severe congestive heart failure. In these patients, the concentration of EDLF immunoreactivity was significantly lower (0.32± 0.09 ng/ml) than in the other 40 patients with AMI and without congestive heart failure (1.51+ 0.32 ng/ml).

Between the period of 24-48 hours after onset of

AMI the plasma levels of EDLF of the AMI group decreased to levels of the control group (0.26± 0.04) , and did not differ significantly from the control values during the subsequent two-week period of assay and observation. Commencing after the secondary of AMI no significant differences were observed in the plasma concentrations of digoxin-like immunoreactivity between patients with uncomplicated AMI and those with AMI complicated by ventricular fibrillation or congestive heart failure.

The results with the human patients discussed in this example were in agreement with the results obtained in Example XIII demonstrating that plasma concentration of the substances having the property to inhibit

Na,K-ATPase is increased in animals exposed to acute coronary ligation. The results of the experimental tests clearly prove proarrhythmic action of EDLF in AMI and the correlation between plasma levels of EDLF and incidence of ventricular arrhythmias, and

antiarrhythmic effect of antidigoxin antibody in

animal models of acute myocardial ischemia.

EXAMPLE XI

Marinobufagin (Mbg) conjugated with a protein, a compound as in Example V, was prepared a follows: 50 mg of marinobufagin, purified by thin layer chromatography, was dissolved in 10ml absolute dry benzene. 80mg of

Ag₂CO₃ was added to the Mbg/benzene solution and heated to boiling with stirring. During the process of

increasing the temperature of the mixture to boiling, a solution of 180mg of acetobromo-D-glucoside in 15ml of dry benzene was added dropwise to the heating

Mbg/benzene/Ag₂CO₃ mixture. Using thin-layer chromatography on silicagel, the reaction was determined to be complete by the disappearance of the spot corresponding to marinobufagin. Following conjugation, the silver salt was filtered followed by evaporation of the

fiItrand in vacuo. The compound is dissolved in ether, filtered and the marinobufagin-glycoside conjugate is crystallized from the filtrand.

EXAMPLE XII

The marinobufagin-glycoside-protein ( BSA) conjugates of the invention were prepared using the method of Example V as used in the conjugation of digoxin with BSA.

EXAMPLE XIII

The monoclonal antibody of the invention was prepared by emulsifying about 1-5 mg/ml of Mbg-BSA conjugate in saline solution with Freund's complete adjuvant 1:1. Emulsification can be readily carried out by repeatedly squirting the suspension through the nozzle of a syringe. A total dosage of about 0.3 ml is injected into multiple sites in mice, for example, in the legs and at the base of the tail. Injections are repeated at intervals of three to five weeks.

Approximately ten days after each treatment, a drop of blood is taken from the tail of each mouse. The

extracted blood is tested for the presence of specific antibodies. The animals yielding the best antiserum are selected for fusion. After a rest period of at least one month, 0.2-0.4 ml of the Mbg-BSA conjugate solution, without Freund's adjuvant, is injected intravenously into each mouse. The injected mice are sacrificed 3-4 days later, the spleens removed unter sterile

conditions. The spleens are placed into a petri dish containing about 5 ml of 2.5% FCS-DMM kept on ice and washed gently. The spleens are transferred to a round- bottomed tube cutting them into three or four pieces per spleen at about 5ml of fresh 2.57. FCS-DMM. Using a Teflon pestle, the pieces are squashed gently to make cell suspensions. The clumps and pieces of connective tissues are allowed to sediment, then the cell

suspensions are transferred to round-bottomed tubes. The tubes are filled with 2.5% FCS-DMM and spun at room temperature for 7-10 minutes at 400g. The

pellets are resuspended in about 10ml of fresh medium and centrifuged as above. The pellets were resuspended in 10 ml of medium, and the cells counted. Viability at this point should be higher than 80%.

Enough myeloma cells from a culture in logarithmic growth are pelleted by centrifugation at room

temperature for 10 minutes at 400g. The pel lets are resuspended in 10ml of 2.57. of FCS-DMM and counted.

Although the fusion and the initial selection of hybrids by growth in HAT medium are quite distinct stages, for convenience they are described together. For

convenience, the fusion of cells in suspension is being described as by the spleens.

The Mbg/spleen cells and the myeloma cells are prepared as above. About 10⁸ spleen cells and 10 myeloma cells are mixed. DMM is added to a volume of 50ml. The cells are spun down at room temperature for 8 mimtes at about 400g. The supernatant is removed with a Pasteur pipette connected to a vacuum line. Complete removal of the supernatant is essential to avoid di luti on of the PEG ( polyethylene glycol

solution) . The pellet is broken by gently tapping the bottom of the tube . The tube is placed in a 200-ml beaker containing water at 40°C and maintained there during the fusion.

0.8 ml of 50% PEG prewarmed at 40% to the pellet using a 1-ml pipette, over a period of 1 minute,

continuously stirring the cells with the pipette tip stirring of the cells in 50% PEG is continued for a further 1.5 minutes. Agglutination of the cells is evident. With the same pipette, 1ml of DMM is added, taken from a tube containing 10ml of DMM kept at 37°C in hot block, to the fusion mixture, continuously stirring as before, over a period of 1 minute. The preceding step is repeated and then repeated twice adding the medium in 30 seconds. Using the same pipette with continuous stirring, add the rest of the 10ml of DMM over a period of about 2 minutes.

With a 10ml pipette add 12-13ml of prewarmed DMM and spin down for about 8 minutes at 400g. The

supernatant is discarded and the pellet gently broken up by tapping the bottom of the tube which is suspended in approximately 49ml of 20% FCS-DMM. This fusion

suspension is distributed in the 48 wells of two Linbro plates. Add a further 1 ml of 20% FCS-DMM . 10⁸ spleen celIs/ml are added to the wells. The wells are incubated overnight at 37°C in a CO₂ incubator.

Using a Pasteur pipette connected to a vacuum line, 1ml of the culture medium is removed from each well without disturbing the cells. The plate is fed with a 1ml HAT medium for 2-3 days afterwards until a vigorous growth of hybrids is evident under the microscope, from 10 days to a month. The culture becomes more yellow and may be tested for antibody activity. Duplicates of the growing hybrid cultures, either all or selected ones, are prepared and fed for a week with HT medium.

While the invention has been described in detail and with reference to specific embodiments thereof, it is apparent to one skilled in the art that various changes and modifications be made therein without departing from the spirit and the scope of the present invention.

Claims

CLAIMS I claim:

Claim 1. The polyclonal antibody raised against marinobufagin which specifically binds marinobufagin.

Claim 2. A polyclonal monospecific antimarinobufagin antibody.

Claim 3. The monoclonal antibody raised against marinobufagin which specifically binds marinobufagin.

Claim 4. An antibody composition consisting of antimarinobufagin antibody in a therapeutically- effective concentration and a non-toxic,

pharmaceutically-acceptable, carrier.

Claim 5. The method for diagnosing cardiac arrhythmias by an increase in the output of

marinobufagin in the ischemic heart of mammal.

Claim 6. The method of treating acute myocardial ischemia in mammal by administering the antibody raised against marinobufagin which specifically binds

marinobufagin to said mammal in admixture with a non-toxic, pharmaceutically-acceptable carrier, said compound or mixture of compounds being in a therapeutically-effective concentration.

Claim 7. The method of treating hypertension in mammal by administering the antibody raised against marinobufagin which specifically binds marinobufagin to said mammal in admixt e with a non-toxic, pharmaceutically-acceptable carrier, said compound or mixture of compounds being in a therapeutically- effective concentration.