CA1051870A - Digoxin derivatives - Google Patents

Abstract

DIGOXIN DERIVATIVES

Abstract of the Disclosure Lysyl tyrosine methyl ester can be coupled to digoxin (digoxigenin tridigitoxoside) by reacting the .epsilon.-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 125I 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 anti-digoxin antibodies about equal to that of unlabelled digoxin, and facilitates the radioimmunoassay of digoxin using gamma scintillation counters.

Description

Background of the Invention Field:
-This invention relates generally to the field of radio assays and specifically to a radioactively labelled digoxin de-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), which methods are based on the use of radioactively labelled substances which can form immunochemical complexes with antibodies specific to that substance. 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 ~J~

amount of the substance tunlabelled) for a limited number of com- _ plexing sites on antibodies specific to the substance. Thus, the RIA of a given substance is performed as follows: a known amount of the substance (labelled) and the unknown amount of the sub-stance (unlabelled) are incubated with anti-substance antibodies.
,~.
During incubation, there are formed immunochemical complexes of k.~
t both antibody-substance (labelled) and antibody-substance (unlabelled). After an appropriate incubation period, 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 ~ ,t"
be used to determine the unknown coDcentration by relating the counts to a standard curve prepared beforehand using known amounts of unlabelled substance.
An essential reagent for the RIA of a given substance is the ,~
1r.,.'_r ~
labelled substance. Ideally, the labelled substance (or a label- ~
~, ~.~r led derivative of the substance, having complexing ability) has . t' an affinity for the anti-substance antibodies which is about equal to the affinity of the unlabelled substance. ~
,';. '.
Prior Art: i ~;''.;. , Digoxin, sometimes described as digoxigenin tridigitoxoside, ~?~
is a cardiac glycoside ~hich 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. Hence, it is extremely important to have a reliable method for accurately determining digoxin concentrations in serum or plasma samples.
To date, RI~ offers the only practical method for determining ~:
serum digoxin concentrations since the clinically significant ~ ~
concentration rarlge of the substance is very ]ow (e.g. approxi-mately 0.5 to about 5 nanogr~ms per milliliter of biological flu;d or scru~).

~ ~ _ . ., _ . , .. ... . _ . _ _ _..... _ _ . . ... . . ...

1051~370 A very common method for labelling di~oxin for use in the RIA of digoxin involves tritiating a sample of digoxin. The tritiation replaces H atoms with 3H atoms on the di@oxin mole-cule ard the replacement can be either random or specific depend-ing on the tritiation method used. Since tritium has a relatively lone radiation half life (e.g. about 12.3 years), 3H-labelled t~.~5 digoxin has the advantage of having a relatively long shelf-life. However, since H is a relatively weak beta radiation emitter, the use of 3H-labelled digoxin commonly requires that added processing steps and materials be used prior to counting ~Y
with a liquid scintillation counter. For example, in using 3H- '.~.',;'!~
labelled digox~in, it is a common practice, after separation of complexed products, to prepare separate cocktails (solutions) containing the labelled substance before the beta radioactivity can be determined. Further, the counts must often be amplified, thus requiring relztively elaborate and sophisticated counting equipment. , ~f~
Because H is a relatively weak emitter of radiation, con- i sideration has been given to labelling digoxin with an isotope of iodine such as 5I which emits a more energetic gamma radia-tion. In using a label such as 5I, however, care must be taken to assure that the label (or tag) is spaced some distance i ~
t ~ b ' away from the antigenic moiety of the digoxin molecule so that the antigenic moiety (or complexing portion) of the digoxin will not be irreparably modified by radiation from the label. If such modification occurs, there can result a loss in affinity of the labelled digoxin for the anti-digoxin antibodies. Such a loss in affinity affects the accuracy and reliability of a ~
RIA of digoxin since an accurate RIA presupposes an equal com- _ petition between labelled and unlabelled digoxin for a limited number of complexin~ sites on anti-digoxln antibodies.

lOS~70 Various techniques for labelling certain steroid-albumin conJugates with 5I for use as tracers in radioimmunoassays are disclosed by S. L. Jeffocate et al. in Clinica Chimica Acta, 43, 343-3~9 (1973). Methods for con~ugating digoxin to the amino groups of lysine residues in human serum albumin are disclosed by T. W. Smith et al., in Biochemistry, 9, No. 2, 331-337 (1970j and by V. P. Bulter et al. in Proc. N.A.S., 57, 71-78 (1967). A method of labelling a digoxin derivative with 5I is disclosed by S. Gutcho et al. in Clin. Chem. 19/,, 1050-59 (1973). In that disclosure, 3-0-succinyl digoxigenin ( 5I) tyrosine is used as the labelled substance and that sub-stance is used in comparative tests with H-digoxin. As dis-closed in that article, however, the affinity of the labelled derivative for anti-digoxin antibodies did not fully correlate with the affinity of unlabelled digoxin.
We have found that a digoxin derivative can be prepared which can be readily labelled with 5I and the labelled deriva-tive 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.

Summary of the Invention Our digoxin derivative consists of the compound formed by reacting the ~-amino group on the lysine residue of lysyl tyro-sine 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 ben~yl ring of the tyrosine residue -can be iodinated with 5I, thereby providing a spaced gamma label and a labellcd digoxin derivative which has an affinity for digoxin antibodies a~out c~ual to digoxill.

~tg D ~`l ~05~870 In one aspect of this invention there is provided a digoxin derivative having the following chemical structure:

I C-N C~H-CH~-CH~-CH~-CH2 --O~C NH2 .~1~

wherein Rl and R2, which may be the same or different, each represents H or I.
In another aspect of this invention there is provided a method of making a digoxin derivative of the formula:

:1 C NH-C-CH-CHz-CHI-CH~-CH~-N~
OeC NH2 I~

comprising the steps of:
(a) reacting digoxin of the formula:

0~ 0~1 ' ~

with-sodium metaperiodate to form a first digoxin derivative of the formula:

~ - 4(a) ~
:,~

~f4,o-(b) reacting the first derivative with lysyl tryosine methyl ester of the formula:

OH

H C-NH-C--CH-CH~-CHI-CH~-CH~-NH~
O-C NH~

CH, ' ~ .

to form a second digoxin derivative of the formula:

~ . H, CH CH, H C-M~-8-CH-CH,-CH,-CH~-CH,-CH~-lFihO~O H

2 0 - O--C NH2 C~ ~ , ~ c) reacting the second digoxin derivative with sodium borohydride to reduce the second digoxin derivative and to form the final digoxin derivative.
In a further aspect of this invention there is provided such a method as described in the immediately preceding paragraph which method further comprises iodinating the tyrosine residue of the final digoxin derivative formed in the step (c) with ~ - ~(b) -. ~ . ~

~7 1 5I ions to form.l25I labelled digoxin derivatives of the formulas: . . rQco ." ~1 ~ CH~o ~O/~

H C-NH-c-cH-cH2-cH2-cH2-cH2 r~HC ~ OOH~ OH
O'C NH2 H~ . .

and H C-NH-C-~H-CH~ H~--CH~-CH~
O~C NH2 .
, I , C~ .

The 125I ions may be obtained from an alkali metal salt of I, more preferably from Na I.

.30 - 4(c) ..

., 1051~37C~
Brief Descriptioll of the Fi~ures FIGS. 1-8 illustrate standard curves generated using our labelled digoxin derivatives which had been stored under vary-ing conditions for various periods of time.
FIG. 9 compares standard curves generated using our label- nnn~r led digoxin derivative and tritiated digoxin. ~
,................................................................................. .,, ~, Specific Embodi~.ents Preparation of Lysyl Tyrosine ~lethyl Ester:

In preparing the product we used L-amino acid residues although it is thought the D-residues could also be successfully used. Our preferred method of preparing the above-described com-pound is as follows: dissolve 2.3 grams of tyrosine methyl ester in 20 ml. of dimethylformamide (DMF) in a 100 ml. round-bottom flask. Stir on a magnetic stirrer. Adjust the pH to 8.o with triethylamine (TEA). Add 5.01 grams of a-t BOC-E-CBZ L-lysine ONP [~-tertiary butyloxy carbonyl e-(carbobenzoxy)-L-lysine ~-nitrophenylester] previously dissolved in-10 ml. of DMF. Mix for 24 hours. ~laintain the pH at 8.o with the TEA. ~hen coupl-ing is complete concentrate off the DMF. The product should form a thic~ gel. Dissolve the product in ethyl acetate (100 .r ~.
ml.). Add 300 m~. of 10~ ammonium hydroxide to the ethyl ace-tate solution. Stir on a magnetic stirred for 30 minutes.
Using a separatory funnel separate the two layers. Wash the ethyl acetate layer with distilled water. Dry the ethyl ace-tate over anhydrous sodium sulfate. Filter away from the sodium sulfate and concentrate the ethyl acetate to an oil. Dissolve the product in cold 100~ trifluoroacetic acid (TFA) and stir for fifteen minutes. Evaporate the TFA to dryness. Dissolve the residue in 15 ml. of glacial acetic acid. Add 15 ml. of cold ~N hydrobro~ic acid and mix for two hours. Add 300 ml. of ethyl ether and mix. A precipitate will appear. Filter auay the pre-cipitate from the filtrate. Place the precipitate under vacuum ~ -for 24 hours. Dissolve the peptide in 10% acetic acid and lyo~ t phllize. The compound has the following structure:
!~
HzC O
H C-NH-C--CH-CHt CHz-CH2-CHz-NHz O~C NH, o CR~

Coupling of Lys~rl ~y~ Methyl Ester to Digoxin:

Digoxin has the following chemical structure:
.' 1 0~} ~~ I '' H O O
OH OH OH
t ~-Cur general method for coupling the lysyl tyrosinè methyl ester to digoxin involves opening the terminal sugar residue on the digoxin with sodium metaperiodate to form: ~r~
.

~o . . ' . ' I

~ _5_ 1 At a pH of 9.0-9.5, the E-~mino group of the lysine residue ¦ _ of the lysyl tyrosine methyl ester is coupled to the terminal ¦
open sugar residue on the digoxin to form:

~ C-1111-C-CH-CH~-CH~-CH, CHI-C~
O~C NH2 H
o , .
CH, ~., With the addition of sodium borohydride, the above compound is ~ .

reduced to: - ¦
~10 CH ,I ~lc H C-~lH-C-CH-CH,-CH,-CH~-CH~-h-HC
O~ HI ' ' !, '`'.

Our actual steps used in preparing the above compound are as follows:
First, 436 m~ (o.56 mole) digoxin is suspended in 20 ml.
of absolute ethanol at room temperature in a 250 ml. Erlenmeyer flask. Then, 20 ml. of 0.~ sodium metaperiodate added. After 25 minutes, O.6 ml. of LM ethylene glycol is added. Five minutes later, the reaction mixture is added to 900 mg of the lysyl tyro-sine methyl ester iD 20 ml. of an aqueous buffer solution which had previously been ad~usted to pH 9.5 with 5% K2C03. The pH is maintained at 9.0-9.5 with the K2C03 for 45 minutes. Then 300 mg of sodium boronydride freshly dissolved in 20 ml. of water, is added. Three hours latcr, the p~ is raised to ~.5 by the addition _7_ Or IM ~m~onium hydroxide. The entire reaction mixture is stirred overni6ht. After the above period the pH is reduced to 4.5 by the addition Or O.lN HCl. After 4 hours at 4C. the entire re-action mixture is centrifu6ed and the precipitate dissolved iD

.
10% ~retic acid and lyophilized. Ihe product is stored at -20C.

Iodinization Or the Digoxin Derivative:

The iodinization is carried out accordin~ to the general procedure suggested by Hunter and Greenwood in Nature, Vol. 194, ~95-6 (1962).
One millicurie of Na 1 5I is reacted with 10-20 ~ gms Or the digoxin derivative in a small vial or tube. chloramine-T is added and quickly mixed. After a-10 seconds sodium metabisulfite (100 ~ gms) is added and mixed also quickly. 500 ~ gms of potas-siu~ iodide is added and mixed The entire reaction mixture is transferred to a DEAE Sephadex column and eluted with O.lM Na phosphate buffer. Three ml. aliquots are collected. Each aliquot is assayed for binding with digoxin antibody. Tubes with hiehest - binding activity are pooled and aliquoted out and stored at appro-priate temperature. In all cases the rea~ents are dissolved in O.lM sodium phosphate pH 7,4.
In follo~ing the above procedure the di6oxin derivative is labelled at C3 or,C3 and C5 Or the benzyl rin6 of the tyrosine residue, depending generally on duration of the iodination pro-cedure. The actu~l perccnta~es Or mono- and di-iodinated deriva-tive can be readily determined by conventional strip scanninG
techniques. Thus, the final iodinated derivative can have either or both Or the followin6 structures:

*Trade Mark t -8-1 10518'7~U
(mono-) HC-hH-~-CH-CYi-C~I-CH~-O-C NH~
o C~3 (di-) 5~ ~f H C^NH-C-CH-CH2-CH2--CHz-CHz-N~O~O~ OH
H OH OH
O=C NH2 CH~
Preparation of Standard Curves:

Samples of the labelled digoxin derivative were used to prepare a series of standard curves which reflected the com-plexing ability of the derivatives after storage at different temperatures and for varying periods of time. These curves are indicated in FIGS. 1-8. All curves were prepared with anti-digoxin antiserum obtained from Biospheres, Inc. of Miami, Florida. The anti-serum had been immobilized by chemical coup-ling through a silane coupling agent to porous glass particles.
The immobilization procedure is described in copending patent application Serial No. 218,435, filed January 22, 1975, en-titled, "Solid Phase Radioimmunoassay", and assigned to the pre-sent assignee. The assay conditions were as follows: a twenty-minute incubation period was followed by a five-minute centri-fugation at 2500 rpm. After centrifugation, the supernatent lOS1870 was removed by aspmration (with no decantation).
Figure 1 illustrates digoxin standard curve using 125I
digoxin that has been stored at 4C. for 2 6 days.
Figure 2 illustrates digoxin curve using 125I digoxin 5 stored at room temperature for 26 days.
Figure 3 illustrates digoxin curve using 125I digoxin stored at 37C. for 26 days.
The above materials were stored in PBS/BSA buffered solu-tions (no NaN3).
Figure 4 illustrates digoxin standard curve using 125I
digoxin that has been frozen for 14 days.
Figure 5 illustrates digoxin curve using 125I digoxln that has been stored at room temperature for 14 days.
Figure 6 illustrates digoxin curve using 125I digoxin 15 that has been stored at room temperature for 14 days.
Figure 7 illustrates digoxin curve using 125 I digoxin curve using 125I digoxin that has been stored at 37C for 14 days.
Figure 8 illustrates digoxin curve using 125I digoxin 20 that has been stored in lyophilized form for 14 days. The buffers used in iodinating the derivatives used in preparing the standard curves of FIGS. 4-8 was PBS/BSA NaN3.

Comparison with other 125I-Digoxin Derivatives and Tritiated Digoxin:

~sing essentially the same assay conditions, our 125I-digoxin derivative was used to prepare a standard curve essen-tially identical to that generated with tritiated digoxin ob-tained from New England Nuclear Corp. The curves are com-pared in FIG. 9 where 125I Tracer designates the curve obtained with our 5I-digoxin derivative and 3~ Trncer repre~ents the curve generated with tritiated digox:in.
In FIG. ~ it can be seen that our labelled digoxin deriva- ,.
tive has an affinity for anti-digoxin antibodies closely ~pprox-imating that of tritiated digoxin.
.' ' ~
.
.,~ ' ' '~

,, ' ' i 'i~
I . . .

~' , ~:~
- . .~ .y,

Claims (8)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined:

1. A digoxin derivative having the following chemical structure:
wherein R1 and R2, which may be the same or different, each represents H or 125I.

2. A digoxin derivative according to Claim 1 wherein R1 represents H and R2 represents 125I.

3. A digoxin derivative according to Claim 1 wherein R1 and R2 both represent 125I.

4. A digoxin derivative according to Claim 1 wherein R1 and R2 both represent H.

5. A method of making a digoxin derivative of the formula:
comprising the steps of:
(a) reacting digoxin of the formula:

with sodium metaperiodate to form a first digoxin derivative of the formula:

(b) reacting the first derivative with lysyl tryosine methyl ester of the formula:
to form a second digoxin derivative of the formula:

(c) reacting the second digoxin derivative with sodium borohydride to reduce the second digoxin derivative and to form the final digoxin derivative.

6. The method according to Claim 5 further comprising iodinating the tyrosine residue of the final digoxin derivatives formed in the step (c) with 125I ions to form 125I labelled digoxin derivatibes of the formulas:

and

7. The method according to Claim 6 wherein the 125I
ions are obtained from an alkali metal salt of 125I.

8. The method according to Claim 7 wherein the alkali metal salt is sodium salt.