Note: Descriptions are shown in the official language in which they were submitted.
M76-D58
(now 77-~0)
Z093
Digoxin, the major cardiac glycoside utilized
for the management of cardiac disorder, imposes a narrow
margin between effective digitalization and toxicity.
Congestive heart failure, whether the result
of absolute or relative cardiac dysfunction, continues to
be the principal indication for therapy with digitalis
glycosides. The degree of improvement in circulatory
dynamics derived from the inotropic action of these agents
is dependent, however, on the setting in which the de-
10 compensation occurs. Thus, the glycosides are most ~`~
bensficial when heart failure results from chronic muscle
malfunction or from certaln pressure and volume overloads
on the ventricles such as in myocardiopathies, athero-
sclerotic heart disease, systemic hypertensio~ and a nwnber
..::. :
of types of congenital heart diseases and acquired valvular
lesions. Digitalis has proven to be less useful when cardiac
decompensation is due to active myocarditis.
Digoxin is excreted primarily by the kidneys
in an unaltered state, and in patients with normal renal
function, the average measured half-time of tritiated
digoxin is 1.6 days. As renal function diminlshes, digoxin ; -
half-time in the body is prolonged. lhe serum digoxin con-
centration and therapeutic effectiveness of digoxin relate
to a number offactors, which include the following: -
25 methodology; post-dose sampling time; dose; potency and -
. i :
bioavailability; malabsorption; abnormal thyroid function;
abnormal metabolism; age, type of heart disease; and renal
fun^tion. ~table serum and myocardial digoxin levels have ~;
been found six hours after the drug's administration.
2 --
.. ..
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:, '' , . , '~
lVl'/ ~ - L)7 0
(now 77~40) ~
, ,
~L0~2~93 ~
The clinical use of digoxin is accompanied by
a distressingly high prevalence of toxic manifestations,
the m~st serious of which a~ arrhythmias and disturbances
of conduction. Many factors such as high dosage, diminished
excretion due to renal disease, old age, and hypothyroidism
contribute to the development of digoxin toxicity. However,
the most important factor is theaccumulation of excessi-ve
amounts of digoxin in the body and in the myocardium in ~
particular. Factors that increase the sensitivity of the -
myocardium to the toxic effects of digitalis are myocardial
ischemia or disease, electrolyte imbalance due to low serum ~- -
potassium or magnesium levels or high serum calcium levels, ~ i
anoxia and alkalosis. ~he~therapeutic margin for digoxin ; ~;
is very narrow (0.5-2.0 ng./ml.). In general, the mean ;
15 digoxin concentrations observed in patients with toxic ~ ~`
manifestations are about 2-fold higher than those of
patients without toxicity. Despite the significantly
different mean levels, however, overlap has been observed,
and it must be emphasized that no arbitrary level can be ~-~
chosen which clearly differentiates toxic from non-toxic
serum digoxin concentrations. It must be rem~mbered that `
digoxin toxicity is the result of interaction of many
factors, each of which deserves individual attention in a
B particular clinical situation.
In accordance with the present invention,
there are disclosed histamine derivatives of digoxigenin
having the following structural formula: ~ -
. . . - .
, .:: . '
.',
. . . .
; _ 3 _
:.
:,
. ,, ., .. , . . , -
M76-D5
(now 77_40)
- ~9Z093
',
CH2CH2NH Z-OY "
//~ ,`' . .
X ~ 4 32 ~ Xl
.~3: ;.
I
.~ ...
wherein X and Xl are the same or different radicals select-
ed from H, 125I or 131I and Z is succinyl, maleoyl, fumaroyl,
:, :
phthaloyl or a radical of the formula~
O Rl R2 O :: .
5~c (C~2)nl _l(CH2)n C
wherein Rl, R2, R3 and R4 are the same or different radicals
selected from hydrogen Or lower alkyl of from 1 to 5 carbon .
B atoms, such as methyl, ethyl, propyl and the like)or Rl and
R may be joined together with tkecarbon atoms to which they
10 are attached to form a cycloalkyl of from 4 to 6 nuclear :
. . . .
carbon atoms such as cyclobutyl, cyclopentyl, cyc}ohexyl and . ~
, . .
the like which may be saturated or unsaturated and which may
be unsubstituted or substituted with various radicals such ~ .
as lower alkyl and the like and n is an integer of O to 2. .
Y is the radical derived from digoxigenin and has the follow- . .
ing s-tructural formula:
.
~'~0 '' ~ ~
~J~
OH `~ `
.~;
-'
, ; , . . . .
M76-D58
(now 77-40)
2093
';'.: ~:
. . .
For the radioimmunoassay of digoxin, the~e -
have been developed various radioiodinated a~ino-acid
derivatives of digoxigenin and of digoxin. See, for
example, U. S. Pat. No. 3,855,208, December 17, 1974, -
Rutner, Rapun and Lewin; Oliver, et al., lhe Journal of
Clinical Investi~atio 1 , 1035-1042 ~1968); U. S. Pat.
No. 3,810,886, May 14, 1974, Rutner, Rapun and Le-~in,
Becton-Dickinson and Company; Smith, Butler and Haber,
New En~land J urnal of Medicine 281, 1212-1216 (1969); U. S.
o Pat. No. 3,92~,355, December 9, 1975, Piasio and Woiszwillo,
Corning Glass Works and German Pat. No. 2,331,922, ;~;
January lO, 1974, The Wellcome Foundation Ltd., LondonO ~-
Previollsly described radioimmunoassays of
digoxin have drawbacks associated with the radio-tracer
15 used. lhe previously described radioimmunoassay which ~`
utilizes tritiated digoxin as a radio-tracer [Smith,
Butler and ~aber, New_En~land Journal of Medicine 281,
1212-1216 (1969)] suffers from drawbacks, i~herent in the
use of tritiated tracers, i.e., low specific activity and
the use of liquid scintillation counting. Previous radio-
immunoassay utilizing radioiodina~ed digoxin or digoxigenin
derivatives as radio-tracer aresensitive to serum varia-
. . . . .
bility [Anggard, et al., New En~land Journal of Medicine 287, -
935 (1972 and Burnett, et al., _ in 1 Chomistry 19, 725
(1973)]. Specifically, the use of radioiodinated digoxigen-
in-3-succinyltyrosine in a radioimmunoassay has been shown
.. . .. ... ...
to produce erroneously high or low values for digoxin
because of inter-sample serum (plasma) variation~ [Cerceo
and Elloso, Clinical Cnemis_ry 18, 539-543 (1972)].
.
-- 5 --
M76-D58
(now 77-~o)
,, .~
1~)9~ 3
~. .
The small difference between the therapeutic `~
and toxic blood levels of digoxin requires an extremely -
-accurate method to measure digoxin levels in serum. This
need is satisfied in principle by radioim~unoassay. Eow-
ever, in order to realize the full potential of radioimmlmo-
assag, a high specific activity X-ray or gamma-ray emitting
radiotracer which is unaffected by variations in sample to
sample composition must be integrated into a simple assay
procedure.
Ihe radioiodinated histamine derivatives of
digoxigenin of the present invention are high specific ac-
tivity X-ray emitters and when used in an RIA procedure
with a gamma-counter, produce reliable ~lantitation of di-
goxin levels in serum and plasma in spite of normal sample ~
to sample variations which might produce erroneous results - -
if other radioiodinated tracers were utilized.
In general, compounds of this invention may
be prepared from digoxigenin by produci~g a hemi-ester of a
dicarboxylic acid with the 3-hydroxy group of digoxigenin,
for example, digoxigenin-3-hemisuccinate, a known compound,
and then reacting the hemi-ester obtained with histamine to
produce the histamine derivative of Formula I in which both
X and Xl are hydrogen; then aither X or Xl, or both, may be
replaced by 125I or 131I
The digoxigenin hemi-esters employed as inter- ,
mediates can be prepared by several known methods including
one which Oliver, et al., [The Journal of Clinical Investi-
gation 47, 1035-1042 (1969)] used to prepare digitoxigenin-
3-hemisuccinate. The acylation reaction employed 'nerein is
an extension of the acylation in the cited work and is
-- 6 --
. . ,, ~ .
.
(now 77-40)
~ z 09 3
well-known to those skilled in the art. I5 must b~ noted
that t~e above met~ods are ~pplicable generally to cyclic
anhydrides, such as glutaric, maleic and phthalic anhydrides.
Saveral of these products obtained ars known in the art.
~ he histamine derivatives of Formula I can bs
prepared from the digoxigenln-3-hamiesters by the following `~
altsrnative three msthods: ~1) by the mixed anhy~ride
procedure described by Erlanger, et al., Journal of Bio-
lo~ical Chemistry 228, 713 (1957); (2) by the water soluble
carbodiimide msthod detailed in the working examples here- `~
inafter, or (3) by the use of N-ethoxycarbonyl-2-ethoxy~
1,2-dihydroquinoline ~EED~) as utiliz~d by Belleau and ~lalek,
JournaL of the Ameri an Chemical Societ~ 90, 1651-2 (1968).
~hese reactions can be carried out in a wide
variety of solvents including water, dimethyl formamide,
tetrahydrofuran and diethylsulfonate an~ at temperatures in
the range of from about -20 to about 60C.
Tne histamine amides of the digoxigenin-3-
hemiesters can be iodinated with any of the various isotopes
Of iodins using one of seve~al me5hods; for example~
Chloramine-L method first described by Hunter and Greellwo~d,
Nature ~ , 495 (1962) or (2) Iodine Monocnloride method of
Ceska, et al., Acta Enlocrinlo~ica 6'~, 111 (1970).
By varying
the iodine to substrate concentration, either one or two
io~ine atoms can be selectively introduced into the imidazole
ring at either the 2 or 4 position or at both the 2 and 4 positions.
It is not n~cessary to know the actual percentages of mono-
and diiodinated material obtained as long as the activity
3o is constant. An-~ isotops of iodine including, for example,
125I or 131I can be utilized in these iodination methods.
' ~, ' .
B - 7 ~
(now 77-40)
: .
~(~92093
lhe following procedures are illustrative of
the present invention
EXAMPLE 1 - Di~oxi~enin-~-succin~lhistamine
A mixture of 500 mg. of digoxigenin-3-hemi-
succinate, 500 mg. of histamine and 500 mg. of 1-ethyl-3-
(3-dimethylaminopropyl)carbodiimide hydrochloride (Ethyl
CDI) in an ethanol/water mixture is stirred at room
temperature for 24 hours. Concentration of the mixture
followed by filtration yields a product which on recrys-
tallization from aqueous ethanol yields substantially pure
digoxigenin-3-succinyl histamine, mOp. 212-214C. (d). ; -
The product shows a single dinitrobenzene positive spot `-
by tlc, U~ analysis shows log E 4.26 at 211 nm; ir; nmr
and nitrogen analysis (Molecular Formula C32X45N307; Calc.
7.2%; Found 7.0%) agree with s-tructure assignment.
EXAMPLE 2 - Di oxigenin-3-succin~1-4' or 2'-125iodohistamine
Iodination of 5 ~g of digoxigenin-3-succinyl-
histamine with 5 mci of 125I is accomplished by the method
of Hunter and Greenwood, Nature 194, 495 (1962). Separation
of product from unreacted iodine and unreacted digoxigenin-
3-succinylhistamine is achieved by passage through a column
of a crosslinked dextr~ gel (G-15SephadexR). Ihin layer
chromatography demonstrates that the purified product~
digoxigenin-3-succinyl-4' or 2'-125iodohistamine, has a
radiochemical purity of greater than 98~.
Varying the iodine to substrate ratio results
in varying proportions of digoxigenin-3-succinyl-4' or 2'-
iodohistamine and digoxigenin-3-succinyl-2', 4'-di-
125iodohistamine being formed.
- '~ , ' '
M'76-~)5~3
(now 77-40)
~ .
'' ' :, ~O~Z093
::
EXAMPLE ~ - Digoxi~eni~n-~-phthaloglhistamine
A mixture of 500 mg. of digoxi;,enin-3-h3mi-
phthalate, 500 mg. of histamine and 500 mg. of 1-ethyl-3-
(3-dimethylaminopropyl)carbodiimide hydrochloride in an
5 ethanol/water mixture is stirred at room temperature for
24 hours. Concentration of the mixture and filtration
affords ~igoxigenin-3-phthaloylhistamine.
EXAMPLE 4 - Di~oxi~enin-~-~hthalo~l-4'- or 2'-125iodohistamine ~
Iodination of 5 g. of digoxigenin-3-phthaloyl- ~ -
10 histamine with 5 mci 125I is accomplished by the method of
Hunter and Greenwood, Nature 194, 495 (1952). The product ` ! .
is separated from unreacted iodine and unreacted digoxigenin-
3-phthaloyl histaaline by passage through a column of a cross-
linked dextran gel to afford digoxigenin-3-phthaloyl-4l or 2'- -
15 125iodohistamine. Varying the iodine to substrate ratio
results in varying proportions of digoxigenin-3-phthaloyl-
4' or 2'-1-25iodohistamine and digoxigenin-3-phthaloyl-2',
4'-dil25iodohistamin3.
~he following equation taken together with
20 Table I illustrate the process of this i~vention and
- additional products which can be produced:
" ' '
" ~;''' ': ' '
;-
.
;:. ': . .
.
_ 9 _
M76-D58.
(now 77-40)
.
~0~31Z~3
HO-Y
Digoxigenin
\ Anhydride ( (Z) )
Ho-Z-O-Y
Histamine~ethyl CDI :
N~ CH2CH2NH-Z-O-Y
Na 1251 (or Na 131I)
. Chloroamine-I ~ .
\ / '' .':' ~:
J.I2C~2N~Z-o-y
. ~:.
.,''`,''
~ / ,',~
~N ~ c:~2C~2N
(13~ 2~I/\N/~125-,[ tl31I) " ~ '. ''
':' .
.,
~ .
- 10 --
.. . .
,
M76-D5~ , :~
(now 77-40) : ~:
gz~
. .
EXAMPLE NO Z ~ ~
~ .
O O - .:,: ,
-CCJ.I=CHC- ~:
O CH O :~:
Il . 1 3 1~ .
6 -cc-il2c~2cE--C-
O O
Il . .
-CCH2C (CH3 ) 2CH2
O O'' ,~'.'": ' '
8 _c n C-
. . .
9 - QC : ~
~C~3 ~ ;~
11 cQc ~ ::
.
~ 1l
: 12 -C C-
' '~
' ' .
~,:,"'
','~''''~ .
- 11 - :
. . . . . ~ ~ ,. : :. . .
M76-D~8
~now 77-40)
2~g3
... ..
The described invention can beutilized in -
various types of radioassays for digoxin. In radioimmuno-
assay (RIA), radiolabelled exogenous antigen competes with `
unlabelled endogenous antigen for binding sites on a specific
antibody. If a high specific activity antigen (Ci/mmole)
is employed, lçss mass of radioactive antigen is needed in
the reastion with the antibody, thereby increasing the
sensitivity of the assay. Determina,,ion of the amount of
radioactivity associated with the antigen-antibody complex
permits quantitative measurement. Because of this unique
affinity reaction between antigen and antibody, in combina-
tion with radioactive tracer analysis, RIA procedures offer
great sensitivity and specificity.
The assay utilizes a treated tube procedure.
8 x 50 mm polypropylene assay tubes are treated with an
antibody to digoxin produced in rabbits. In this assay,
radioiodinated digoxin and digoxin from the patient samples
are incubated in the treated tube. Labelled and endogenous
digoxin compete for binding sites on the antibody. The
antigen-antibody complex is separated from free antigen by
aspiration and washing of the assa-; tube. The quantity of
radioactivity remaining bound to the assay tube is compared
with th3 value for known digoxin standards. Digoxin in the
patient sample can then be calculatedO A reliable radio-
immunoassay procedure utilizing the product prepared in
- Example 2 is as follows:
A method for measuring the digoxin content of
a serum sa~ple comprises (a) adding to antibody coated tubes
a mixture of a tracer amount of a radiolabelled histamine- ;
derivative of digoxigenin and a serum sample; (b) incubating
said mixture to pérmit the digoxin in the sample an~said
. ~.
- 12
M~ d
(now 77-40)
- : .
~ ~09Z~393 : ~
radiolabelled derivative to bind the digoxin antibody; (c)
. .
scparating the bound labelled digoxln from the fres digoxin
and (d) measuring the radioac-tivity.
A detailed description of the assay is as
follows:
Serum (20 ~1) microlitersof unknown digoxin
content and four more standards containing a known amount
of digoxin and 980,~1 of assay buffer (phosphate buffered
saline at p~ 7) containing 2 pg of the radioiodinated
derivative of this invention are added to plastic tubes
coated with digoxin specific antibodies [Science 1~8, 1570
(1967)]. The mixture of each tube is vortexed and then
incubaked for two hours at 37C, The antibody coated tubes
are th~n aspirated and washed two times with distilled
water. Competition betweeen endogenous digoxin an~ the
radioiodinated digoxigenin product of Example 2 for anti~
body combining sites determines the amount of radioiodin-
- .
ated compound whlch becomes bound to the antibody coated
tube. The washed antib3dy coated tubes are counted for
residual X-ray radiation and the values (counts) obtained
from the standards are used to plot a standard curve. The
value obtained for the serum is plotted on the standard
curve ko determine the level of digoxin in the tested serum.
~'' ~, :
''''" "."~ .
- 13 -
. . , , .. : .
. ~ . . . .
.~ , . . . . .
. :: , . . .
