US3925355A - Labelled digoxin derivatives for radioimmunoassay - Google Patents

Labelled digoxin derivatives for radioimmunoassay Download PDF

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Publication number
US3925355A
US3925355A US447249A US44724974A US3925355A US 3925355 A US3925355 A US 3925355A US 447249 A US447249 A US 447249A US 44724974 A US44724974 A US 44724974A US 3925355 A US3925355 A US 3925355A
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Prior art keywords
digoxin
derivative
labelled
radioimmunoassay
substance
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Expired - Lifetime
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US447249A
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English (en)
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Roger N Piasio
James E Woiszwillo
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Bayer Corp
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Corning Glass Works
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Priority to US447249A priority Critical patent/US3925355A/en
Priority to DE19752505267 priority patent/DE2505267A1/de
Priority to GB800775A priority patent/GB1453583A/en
Priority to FR7506110A priority patent/FR2262670B1/fr
Priority to JP50024478A priority patent/JPS50117766A/ja
Priority to CA221,002A priority patent/CA1051870A/en
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Publication of US3925355A publication Critical patent/US3925355A/en
Assigned to CIBA CORNING DIAGNOSTICS CORP., A CORP. OF DE. reassignment CIBA CORNING DIAGNOSTICS CORP., A CORP. OF DE. ASSIGNMENT OF ASSIGNORS INTEREST. Assignors: CORNING GLASS WORKS, A BUSINESS CORP. OF NEW YORK
Assigned to CIBA CORNING DIAGNOSTICS CORP., A CORP. OF DE. reassignment CIBA CORNING DIAGNOSTICS CORP., A CORP. OF DE. ASSIGNMENT OF ASSIGNORS INTEREST. Assignors: CORNING GLASS WORKS
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J43/00—Normal steroids having a nitrogen-containing hetero ring spiro-condensed or not condensed with the cyclopenta(a)hydrophenanthrene skeleton
    • C07J43/003—Normal steroids having a nitrogen-containing hetero ring spiro-condensed or not condensed with the cyclopenta(a)hydrophenanthrene skeleton not condensed

Definitions

  • ABSTRACT Lysyl tyrosine methyl ester can be coupled to digoxin (digoxigenin tridigitoxoside) by reacting the e-amino group of the lysine residue with the opened terminal sugar group of digoxin to yield a new digoxin derivative.
  • the derivative can be labelled at the benzyl ring of the tyrosine residue with i to space the label away from the antigenic moiety of the digoxin derivative.
  • the spaced label minimizes deleterious effects of gamma radiation on the antigenic moiety, yields a labelled digoxin derivative having an affinity for antidigoxin antibodies about equal to that of unlabelled digoxin, and facilitates the radioimmunoassay of digoxin using gamma scintillation counters.
  • This invention relates generally to the field of radio assays and specifically to a radioactively labelled digoxin derivative useful in the radioimmunoassay (RIA) of digoxin.
  • Radioimmunoassay is a term used to describe any of several methods for determining very small concentrations of substances (especially in biological fluids),
  • the RIA of a substance for which there exists antibodies is based on the observation that a known amount of that substance (which has been radioactively labelled) will tend to compete equally with the unknown amount of the substance (unlabelled) for a limited number of complexing sites on antibodies specific to the substance.
  • the RIA of a given substance is performed as follows: a known amount of the substance (labelled) and the unknown amount of the substance (unlabelled) are incubated with anti-substance antibodies. During incubation, there are formed immunochemical complexes of both antibody-substance (labelled) and antibody-substance (unlabelled).
  • the immunochemical complexes are removed from the reaction solution. Then, radioactivity measurements (counts) are taken of either the removed complexes or the remaining solution. The counts can then be used to determine the unknown concentration by relating the counts to a standard curve prepared beforehand using known amounts of unlabelled substance.
  • the labelled substance An essential reagent for the RIA of a given substance is the labelled substance.
  • the labelled substance (or a labelled derivative of the substance, having complexing ability) has an affinity for the anti-substance antibodies which is about equal to the affinity of the unlabelled substance.
  • Prior Art Digoxin sometimes described as diogoxigenin tridigitoxoside, is a cardiac glycoside which is used as a heart stimulant.
  • the compound is used in very small amounts and the difference between therapeutic and toxic amounts is very slight.
  • RIA offers the only practical method for determining serum digoxin concentrations since the clinically significant concentration range of the substance is very low (e.g. approximately 0.5 to about 5 nanograms per milliliter of biological fluid or serum).
  • a very common method for labelling digoxin for use in the RIA of digoxin involves tritiating a sample of digoxin.
  • the tritiation replaces H atoms with H atoms on the digoxin molecule and the replacement can be either random or specific depending on the tritiation method used.
  • tritium has a relatively long radiation half life (e.g. about 12.3 years)
  • I l-labelled digoxin has the advantage of having a relatively long shelf-life.
  • H is a relatively weak beta radiation emitter
  • the use of H-labelled digoxin commonly requires that added processing steps and materials be used prior to counting with a liquid scintillation counter.
  • 3-O- succinyl digoxigenin 1) tyrosine is used as the labelled substance and that substance is used in comparative tests with H-digoxin.
  • the affinity of the labelled derivative for antidigoxin antibodies did not fully correlate with the affinity of unlabelled digoxin.
  • a digoxin derivative can be prepared which can be readily labelled with 'I and the labelled derivative has an affinity for anti-digoxin antibodies about equal to that of unlabelled and tritiated digoxin.
  • the derivative and methods for preparing, labelling, and using it are described more fully hereunder.
  • digoxin derivative consists of the compound formed by reacting the e-amino group on the lysine residue of lysyl tyrosine methyl ester with the opened terminal sugar residue of digoxin under reaction conditions described in detail hereunder. Once the lysyl tyrosine methyl ester is coupled through the terminal sugar residue, the benzyl ring of the tyrosine residue can be iodinated with thereby providing a spaced gamma label and a labelled digoxin derivative which has an affinity for digoxin antibodies about equal to digoxin.
  • FIGS. 1-8 illustrate standard curves generated using our labelled digoxin derivatives which had been stored under varying conditions for various periods of time.
  • FIG. 9 compares standard curves generated using our labelled digoxin derivative and tritiated digoxin.
  • Digoxin has the following chemical structure:
  • the e-amino group of the lysine residue of the lysyl tyrosine methyl ester is coupled to the terminal open sugar residue on the digoxin to form:
  • the iodinization is carried out according to the general procedure suggested by Hunter and Greenwood in Nature, Vol. 194, 495-6 (1962).
  • One millicurie of Na I is reacted with 10-20 p. gms of the digoxin derivative in a small vial or tube. Chloramine T is added and quickly mixed. After 8-10 seconds sodium metabisulfite (100 ,u. gms) is added and mixed also quickly. 500 p. gms of potassium iodide is added and mixed. The entire reaction mixture is transferred to a DEAE Sephadex column and eluted with 0.1M Na phosphate buffer. Three ml. aliquots are collected. Each aliquot is assayed for binding with digoxin antibody. Tubes with highest binding activity are pooled and aliquoted out and stored at appropriate temperature. In all cases the reagents are dissolved in 0.1M sodium phosphate pH 7.4.
  • the digoxin derivative is labelled at C or C and C of the benzyl ring of the tyrosine residue, depending generally on duration of the iodination procedure.
  • the actual percentages of monoand di-iodinated derivative can be readily determined by conventional strip scanning techniques.
  • the assay conditions were as follows: a -minute incubation period was followed by a 5-minute centrifugation at 2,500 rpm. After centrifugation, the supernatent was removed by aspiration (with no decantation).
  • FIG. 1 illustrates digoxin standard curve using 1 digoxin that has been stored at 4C. for 26 days.
  • FIG. 2 illustrates digoxin curve using I digoxin l0 stored at room temperature for 26 days.
  • FIG. 7 illustrates digoxin curve using digoxin curve using I digoxin that has been stored at 37C. for 14 days.
  • FIG. 8 illustrates digoxin curve using I digoxin that the final iodinated derivative can have either or both of 25 has been stored in lyophilized form for 14 days.
  • digoxin derivative a digoxin derivative having the following chemical tion of sodium borohydride to reduce the second structure: digoxin derivative.
  • the method of claim 4 which includes the addistructure: tional and subsequent step of iodinating the tyrosine 30 (:H OH

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Steroid Compounds (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
US447249A 1974-03-01 1974-03-01 Labelled digoxin derivatives for radioimmunoassay Expired - Lifetime US3925355A (en)

Priority Applications (6)

Application Number Priority Date Filing Date Title
US447249A US3925355A (en) 1974-03-01 1974-03-01 Labelled digoxin derivatives for radioimmunoassay
DE19752505267 DE2505267A1 (de) 1974-03-01 1975-02-07 Digoxinderivate
GB800775A GB1453583A (en) 1974-03-01 1975-02-26 Digoxin derivatives
JP50024478A JPS50117766A (pl) 1974-03-01 1975-02-27
FR7506110A FR2262670B1 (pl) 1974-03-01 1975-02-27
CA221,002A CA1051870A (en) 1974-03-01 1975-02-28 Digoxin derivatives

Applications Claiming Priority (1)

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US447249A US3925355A (en) 1974-03-01 1974-03-01 Labelled digoxin derivatives for radioimmunoassay

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US3925355A true US3925355A (en) 1975-12-09

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US447249A Expired - Lifetime US3925355A (en) 1974-03-01 1974-03-01 Labelled digoxin derivatives for radioimmunoassay

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US (1) US3925355A (pl)
JP (1) JPS50117766A (pl)
CA (1) CA1051870A (pl)
DE (1) DE2505267A1 (pl)
FR (1) FR2262670B1 (pl)
GB (1) GB1453583A (pl)

Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4029880A (en) * 1974-06-20 1977-06-14 The Radiochemical Centre Limited Labelled derivatives of steroids
US4056608A (en) * 1976-04-08 1977-11-01 Syva Company Cardiac glycoside or aglycone assays
US4064227A (en) * 1975-03-17 1977-12-20 Mallinckrodt, Inc. Radioimmunoassay method for the determination of cardiotonic glycosides
US4184037A (en) * 1973-06-21 1980-01-15 Burroughs Wellcome Co. Digoxin oxidized product
US4202874A (en) * 1976-09-29 1980-05-13 Becton Dickinson & Company Monoradioiodinated derivatives and precursors for production thereon
US4221725A (en) * 1977-08-12 1980-09-09 E. R. Squibb & Sons, Inc. Steroid derivatives and their use in radioimmunoassays
US4230621A (en) * 1978-05-01 1980-10-28 E. R. Squibb & Sons, Inc. Steroid derivatives and their use in radioimmunoassays
US4336185A (en) * 1976-03-02 1982-06-22 Rohm And Haas Company Folic acid derivatives
US4345096A (en) * 1979-01-17 1982-08-17 E. R. Squibb & Sons, Inc. Steroid derivatives and their use in radioimmunoassays
US5104637A (en) * 1985-02-06 1992-04-14 University Of Cincinnati Radio labeled dihematophorphyrin ether and its use in detecting and treating neoplastic tissue
US5198537A (en) * 1988-10-27 1993-03-30 Boehringer Mannheim Gmbh Digoxigenin derivatives and use thereof
WO2003075010A2 (fr) * 2002-03-05 2003-09-12 Centre National De La Recherche Scientifique -Cnrs- Precurseurs de tracteurs immunologiques non-peptidiques comprenant un motif tyrosyl-(x)n -lysine, ou lysine-(x)n -tyrosine
WO2004052913A1 (en) * 2002-12-12 2004-06-24 Bio-Med Reagents Limited A digoxin labelled glycan probe, its use and methods for its production
US20180207189A1 (en) * 2015-07-19 2018-07-26 Yeda Research And Development Co., Ltd. SELECTIVE INHIBITORS OF Alpha2-CONTAINING ISOFORMS OF Na,K-ATPase AND USE THEREOF FOR REDUCTION OF INTRAOCULAR PRESSURE

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4670406A (en) * 1984-01-06 1987-06-02 Becton Dickinson And Company Tracers for use in assays
US4595656A (en) * 1984-01-06 1986-06-17 Becton Dickinson & Company Coupling agents and products produced therefrom
DE3802060A1 (de) * 1988-01-25 1989-07-27 Boehringer Mannheim Gmbh Hapten-protein-konjugate und ihre verwendung
ES2702674T3 (es) 2013-08-29 2019-03-04 Yeda Res & Dev Inhibidores selectivos de la isoforma alfa2 de Na,K-ATPasa y el uso para la reducción de la presión intraocular
WO2017013637A1 (en) * 2015-07-19 2017-01-26 Yeda Research And Development Co. Ltd. Selective inhibitors of alpha2-containing isoforms of na,k-atpase and use thereof for reduction of intraocular pressure

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US2885394A (en) * 1956-12-11 1959-05-05 Lasdon Foundation Inc Modified saccharide compounds
US3014026A (en) * 1958-09-05 1961-12-19 Kroll Harry Chelates of monosaccharide-amino acids and related compounds

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US2885394A (en) * 1956-12-11 1959-05-05 Lasdon Foundation Inc Modified saccharide compounds
US3014026A (en) * 1958-09-05 1961-12-19 Kroll Harry Chelates of monosaccharide-amino acids and related compounds

Cited By (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4184037A (en) * 1973-06-21 1980-01-15 Burroughs Wellcome Co. Digoxin oxidized product
US4029880A (en) * 1974-06-20 1977-06-14 The Radiochemical Centre Limited Labelled derivatives of steroids
US4064227A (en) * 1975-03-17 1977-12-20 Mallinckrodt, Inc. Radioimmunoassay method for the determination of cardiotonic glycosides
US4336185A (en) * 1976-03-02 1982-06-22 Rohm And Haas Company Folic acid derivatives
US4056608A (en) * 1976-04-08 1977-11-01 Syva Company Cardiac glycoside or aglycone assays
US4202874A (en) * 1976-09-29 1980-05-13 Becton Dickinson & Company Monoradioiodinated derivatives and precursors for production thereon
US4221725A (en) * 1977-08-12 1980-09-09 E. R. Squibb & Sons, Inc. Steroid derivatives and their use in radioimmunoassays
US4230621A (en) * 1978-05-01 1980-10-28 E. R. Squibb & Sons, Inc. Steroid derivatives and their use in radioimmunoassays
US4345096A (en) * 1979-01-17 1982-08-17 E. R. Squibb & Sons, Inc. Steroid derivatives and their use in radioimmunoassays
US5104637A (en) * 1985-02-06 1992-04-14 University Of Cincinnati Radio labeled dihematophorphyrin ether and its use in detecting and treating neoplastic tissue
US5198537A (en) * 1988-10-27 1993-03-30 Boehringer Mannheim Gmbh Digoxigenin derivatives and use thereof
WO2003075010A2 (fr) * 2002-03-05 2003-09-12 Centre National De La Recherche Scientifique -Cnrs- Precurseurs de tracteurs immunologiques non-peptidiques comprenant un motif tyrosyl-(x)n -lysine, ou lysine-(x)n -tyrosine
FR2836996A1 (fr) * 2002-03-05 2003-09-12 Centre Nat Rech Scient PRECURSEURS DE TRACEURS IMMUNOLOGIQUES NON-PEPTIDIQUES COMPRENANT UN MOTIF TYROSYL-(X)n-LYSINE-(X)n-TYROSINE, PROCEDE DE PREPARATION ET LEURS APPLICATIONS
WO2003075010A3 (fr) * 2002-03-05 2004-05-06 Centre Nat Rech Scient Precurseurs de tracteurs immunologiques non-peptidiques comprenant un motif tyrosyl-(x)n -lysine, ou lysine-(x)n -tyrosine
WO2004052913A1 (en) * 2002-12-12 2004-06-24 Bio-Med Reagents Limited A digoxin labelled glycan probe, its use and methods for its production
US20180207189A1 (en) * 2015-07-19 2018-07-26 Yeda Research And Development Co., Ltd. SELECTIVE INHIBITORS OF Alpha2-CONTAINING ISOFORMS OF Na,K-ATPase AND USE THEREOF FOR REDUCTION OF INTRAOCULAR PRESSURE
US10668094B2 (en) * 2015-07-19 2020-06-02 Yeda Research And Development Co. Ltd. Selective inhibitors of Alpha2-containing isoforms of Na,K-ATPase and use thereof for reduction of intraocular pressure
US11077128B2 (en) 2015-07-19 2021-08-03 Yeda Research And Development Co. Ltd. Selective inhibitors of Alpha2-containing isoforms of Na,K-ATPase and use thereof for reduction of intraocular pressure

Also Published As

Publication number Publication date
FR2262670B1 (pl) 1978-04-21
GB1453583A (en) 1976-10-27
DE2505267A1 (de) 1975-09-04
JPS50117766A (pl) 1975-09-16
FR2262670A1 (pl) 1975-09-26
CA1051870A (en) 1979-04-03

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Legal Events

Date Code Title Description
AS Assignment

Owner name: CIBA CORNING DIAGNOSTICS CORP., MEDFIELD, MASSACHU

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST.;ASSIGNOR:CORNING GLASS WORKS, A BUSINESS CORP. OF NEW YORK;REEL/FRAME:004480/0063

Effective date: 19851115

AS Assignment

Owner name: CIBA CORNING DIAGNOSTICS CORP., MEDFIELD, MASSACHU

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST.;ASSIGNOR:CORNING GLASS WORKS;REEL/FRAME:004483/0427

Effective date: 19851105