WO1994012210A1 - Anticorps anti-facteurs de type digoxine endogene, composition contenant cet anticorps et procede de diagnostic et de traitement des arythmies cardiaques et de l'hypertension - Google Patents

Anticorps anti-facteurs de type digoxine endogene, composition contenant cet anticorps et procede de diagnostic et de traitement des arythmies cardiaques et de l'hypertension Download PDF

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Publication number
WO1994012210A1
WO1994012210A1 PCT/US1993/011365 US9311365W WO9412210A1 WO 1994012210 A1 WO1994012210 A1 WO 1994012210A1 US 9311365 W US9311365 W US 9311365W WO 9412210 A1 WO9412210 A1 WO 9412210A1
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WO
WIPO (PCT)
Prior art keywords
marinobufagin
edlf
antibody
hypertension
digoxin
Prior art date
Application number
PCT/US1993/011365
Other languages
English (en)
Inventor
Alexei Y. Bagrov
Original Assignee
Bagrov Alexei Y
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Bagrov Alexei Y filed Critical Bagrov Alexei Y
Priority to AU57284/94A priority Critical patent/AU5728494A/en
Publication of WO1994012210A1 publication Critical patent/WO1994012210A1/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J19/00—Normal steroids containing carbon, hydrogen, halogen or oxygen, substituted in position 17 by a lactone ring
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J71/00—Steroids in which the cyclopenta(a)hydrophenanthrene skeleton is condensed with a heterocyclic ring
    • C07J71/0005—Oxygen-containing hetero ring
    • C07J71/001—Oxiranes
    • C07J71/0021—Oxiranes at position 14(15)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • 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
  • invention is further directed to antibodies prepared from steroid compounds derived from the venom and tissues of the Bufo marinus toad for blocking
  • 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
  • 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
  • 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
  • Na + ,K + -ATPase in a manner similar to the digitalis glycosides.
  • 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.
  • AMI Acute myocardial ischemia
  • 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
  • arrhythmias utilizing the antibody of the invention may be by any of the conventional routes of administration, for example, oral, intramuscular, intravenous or rectally.
  • the antibody is preferably administered in combination with a
  • pharmaceutically-acceptable carrier which may be solid or liquid, dependant upon choice and route of
  • acceptable carriers include, but are not limited to, for example,
  • 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
  • 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
  • Antidigoxin immunoglobulin was separated by
  • the left coronary arteries were ligated 1-2 mm distal to their origins.
  • the hearts were
  • 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 ⁇ M phenylmethylsulfonylfluoride (50 ⁇ 1 per 4 ml blood). The resulting solution was frozen at -20°C for determination of digoxin-like immunoreactivity
  • DELFIA enhanced lanthanide fluoroimmuniassay
  • 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:
  • DIGIBIND Fab fragments of bovine antidigoxin antibody, a drug produced for the treatment of digoxin overdose by Burroughs Wellcome Co.
  • EDLF digoxin-like immunoreactivity
  • 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 . ,
  • 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.
  • Digibind has a very high
  • 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. Russia, Russia and Riga, Norway
  • 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.
  • 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,
  • steroids was performed in UV. A spot corresponding to marinobufin was scraped, divided into three (3) portions and extracted with ethyl acetate.
  • 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.
  • Example III 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 CH 3 COONa-CH 3 COOH, 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 Na 2 HPO 4 , 1.5mM KH z PO 4 , 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 (NH 4 ) 2 SO 4 .
  • a phosphate buffer solution 150mM NaCl, 3mM KC1, 8mM Na 2 HPO 4 , 1.5mM KH z PO 4 , 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 (NH 4 ) 2 SO 4 .
  • the phosphate buffer solution 150mM NaCl, 3mM KC1, 8mM Na 2 HPO 4 , 1.5mM KH z PO 4 , pH 7.2.
  • the pH was adjusted to 7.4 with
  • Step 4 The precipitate from Step 3 was
  • 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.
  • 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.
  • 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 ⁇ 1 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
  • 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.
  • 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.
  • 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
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • 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
  • the Mbg/spleen cells and the myeloma cells are prepared as above. About 10 8 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)
  • 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.

Abstract

Cette invention concerne un nouveau composé et de nouvelles compositions d'anticorps utilisés pour prévenir et traiter l'hypertension et les arythmies cardiaques, des procédés de préparation et de diagnostic et/ou de prédiction de l'apparition d'arythmies cardiaques induites par divers états pathologiques. Cette invention concerne également des anticorps préparés à partir de composés stéroïdiens provenant du venin et des tissus du crapaud Bufo marinus qui servent à bloquer les facteurs du type digoxine endogène trouvés dans le plasma de mammifère.
PCT/US1993/011365 1992-12-02 1993-11-29 Anticorps anti-facteurs de type digoxine endogene, composition contenant cet anticorps et procede de diagnostic et de traitement des arythmies cardiaques et de l'hypertension WO1994012210A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU57284/94A AU5728494A (en) 1992-12-02 1993-11-29 Anti-edlf antibody, composition thereof and method of diagnosing and treating cardiac arrhythmias, hypertension

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US98448092A 1992-12-02 1992-12-02
US07/984,480 1992-12-02

Publications (1)

Publication Number Publication Date
WO1994012210A1 true WO1994012210A1 (fr) 1994-06-09

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997024366A1 (fr) * 1995-12-29 1997-07-10 Philip James Hilton Composes inhibiteurs de la pompe a sodium et leurs partenaires de fixation
US5770376A (en) * 1992-12-02 1998-06-23 Biomedical Sciences Research Laboratories, Inc. Method of diagnosing and treating myocardial infarction and hypertension
WO2004071273A2 (fr) 2003-02-04 2004-08-26 The Administrators Of The Tulane Educational Fund Procede pour utiliser l'augmentation de marinobufagenine afin de determiner une preeclampsie et appareil correspondant
CN103439485A (zh) * 2013-08-22 2013-12-11 天津市康婷生物工程有限公司 高效检测内源性洋地黄样因子含量的试剂盒及使用方法
US20160299130A1 (en) * 2015-04-09 2016-10-13 Jules B. Puschett Telocinobufagin (TCINO) in the Diagnosis and Pathogenesis of Preeclampsia, Traumatic Brain Injury and Acute Respiratory Distress Syndrome
EP3449977A1 (fr) 2013-03-15 2019-03-06 Velo Bio, LLC Procédé de traitement de l'éclampsie et de la prééclampsie

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
CHEMICAL PHARMACOLOGY BULLETIN, Volume 34, issued August 1986, K. SHIMADA et al., "Occurrence of Bufadienolides in the Skin of Bufo Viridis LAUR", see pages 3454-3457. *
EUROPEAN JOURNAL OF PHARMACOLOGY, Volume 162, issued September 1989, A.Y. BAGROV et al., "Antiarrhythmic Effect of Antibodies to Digoxin in Acute Myocardial Ischemia in Rats", see pages 195-196. *
J.W. GODING, "Monoclonal Antibodies: Principles and Practices", published 1986, by ACADEMIC PRESS, see pages 59-103 and 281-293. *
LIFE SCIENCES, Volume 35, issued January 1984, C.T. HUANG et al., "Lowering of Blood Pressure in Chronic Aortic Coarctate Hypertensive Rats with Anti-Digoxin Antiserum", pages 115-118. *
NATURE, Volume 274, issued 20 July 1978, J.S FLIER, "Ouabain-Like Activity in Toad Skin and its Implication for Endogenous Regulation of Ion Transport", pages 285-286. *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5770376A (en) * 1992-12-02 1998-06-23 Biomedical Sciences Research Laboratories, Inc. Method of diagnosing and treating myocardial infarction and hypertension
WO1997024366A1 (fr) * 1995-12-29 1997-07-10 Philip James Hilton Composes inhibiteurs de la pompe a sodium et leurs partenaires de fixation
WO2004071273A2 (fr) 2003-02-04 2004-08-26 The Administrators Of The Tulane Educational Fund Procede pour utiliser l'augmentation de marinobufagenine afin de determiner une preeclampsie et appareil correspondant
EP1592967A2 (fr) * 2003-02-04 2005-11-09 The Administrators Of The Tulane University Educational Fund Procede pour utiliser l'augmentation de marinobufagenine afin de determiner une preeclampsie et appareil correspondant
EP3449977A1 (fr) 2013-03-15 2019-03-06 Velo Bio, LLC Procédé de traitement de l'éclampsie et de la prééclampsie
CN103439485A (zh) * 2013-08-22 2013-12-11 天津市康婷生物工程有限公司 高效检测内源性洋地黄样因子含量的试剂盒及使用方法
US20160299130A1 (en) * 2015-04-09 2016-10-13 Jules B. Puschett Telocinobufagin (TCINO) in the Diagnosis and Pathogenesis of Preeclampsia, Traumatic Brain Injury and Acute Respiratory Distress Syndrome

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