Reversal of Advanced Digitoxin Toxicity and
Modification of Pharmacokinetics by Specific
Antibodies and Fab Fragments
Hermann R. Ochs, Thomas W. Smith
J Clin Invest. 1977;60(6):1303-1313. https://doi.org/10.1172/JCI108889.
The effects of Fab fragments of high-affinity specific antibodies have been studied in a canine experimental model of lethal digitoxin toxicity. Selected antiserum from sheep immunized and boosted with a digoxin-serum albumin conjugate contained antibodies that cross-reacted with digitoxin with an average intrinsic association constant of 1.4 × 1010 M−1 as determined by equilibrium dialysis. Rapid second-order association kinetics (kf = 3.7 × 106 M−1 per s) and slow dissociation kinetics (kr = 1.9 × 10−4 per s) were documented for the antibody-digitoxin complex. Eight dogs given 0.5 mg/kg digitoxin intravenously
developed ventricular tachycardia after 23±4 (SEM) min. Control nonspecific Fab fragments were then given. All animals died an average of 101±36 min after digitoxin administration. Another eight dogs given the same digitoxin dose similarly developed ventricular
tachycardia after 28±3 min. This group then received a molar equivalent dose of specific Fab fragments intravenously over 3 min, followed by a 30-min infusion of one-third of the initial dose. All dogs survived. Conducted sinus beats reappeared 18±4 min after initial Fab infusion, and stable normal sinus rhythm was present at 54±16 min. Plasma total digitoxin concentrations increased threefold during the hour after initial Fab infusion, while plasma free digitoxin concentration decreased to less than 0.1 ng/ml. Effects on digitoxin
pharmacokinetics of these Fab fragments and the antibody population from which they […]
Research Article
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Reversal of Advanced
Digitoxin Toxicity
and
Modification of Pharmacokinetics by
Specific Antibodies and Fab Fragments
HERMANN R. OCHSandTHOMAS W. SMITH, CardiovascularDivision, Department of Medicine, PeterBent Brigham Hospital, andthe
Department ofMedicine, Harvard Medical School, Boston,
Massachusetts 02115
A B STR A C T The effects of Fab fragments of high-affinity specific antibodies have been studied in a canineexperimental model of lethal digitoxin toxicity. Selected antiserum from sheep immunized and
boosted withadigoxin-serum albuminconjugate
con-tained antibodies that cross-reacted with digitoxin with an average intrinsic association constant of 1.4 x 10"0 M-1 as determined by equilibrium dialysis. Rapid second-order association kinetics
(kf
=3.7 x 106 M-1 per s) and slow dissociation kinetics(kr
= 1.9 X 10-4 pers)
were documented for theantibody-digitoxin complex. Eight dogs given 0.5 mg/ kg digitoxin intravenously developed ventricular tachycardia after 23±4 (SEM)min.Controlnonspecific Fab fragments were then given. All animals died an
average of101+36 min after digitoxin administration. Another eight dogs giventhesamedigitoxin dose
simi-larly developed ventricular tachycardia after28+3min.
This group then received a molar equivalent dose of specific Fab fragments intravenously over 3 min, fol-lowed by a30-min infusion of one-third of the initial dose. All dogs survived. Conducted sinus beats reap-peared 18+4minafter initial Fab infusion, and stable normalsinusrhythmwaspresentat 54±16min.Plasma total digitoxinconcentrations increased threefold dur-ing the hour after initial Fab infusion, while plasma free digitoxin concentration decreased toless than 0.1
ng/ml. Effects on digitoxin pharmacokinetics ofthese
Fabfragments and the antibody population fromwhich
they were derived were further investigated in a
primate species. Unlike common laboratory animals previously studied, the rhesus monkey was found to
haveaprolongedeliminationhalf-life, estimatedat135
Dr.Ochs was aFellow ofDeutscheForschungsgemeinschaft.
Receivedforpublication20January 1977andin revised form5July1977.
and 118 h by radioimmunoassay and [3H]digitoxin measurements, respectively, similar to man and thus providing a clinically relevant experimental model. Intravenous administration of 2 mol of specific Fab fragments per mole of digitoxin 6 h after 0.2 mg of digitoxin producedarapid 4.3-fold increaseinplasma total digitoxin concentration followed by a rapid fall
(t1 4 h) accompanied by a 14-fold enhancement of urinary digitoxin excretion overcontrol values during the 6-hperiod after Fabwas given. Analytical studies wereconsistent with increased excretion ofnative digi-toxin rather than metabolites, and the glycoside was
found in equilibrium dialysis studies to be excreted in the urine in Fab-bound form. Administration of2
mol of specific antibody binding sites per mole of digi-toxin as intact IgG caused a greater and more pro-longed increase in plasma total digitoxin concentra-tion, peaking 13-fold above control levels. Incontrast
to the effects of Fab, however, specific IgG reduced
the rate of urinary digitoxin excretion substantially below control values. We conclude that Fab fragments of antibodies with high affinity for digitoxin are ca-pable of rapid reversal of advanced, otherwise lethal digitoxin toxicity, and are capable of reducing the plasma half-life and accelerating urinary excretion of digitoxin.
INTRODUCTION
Severe digitalis toxicity resistant to conventional therapy remains an important clinical problem (1, 2). Substantial experimental literature now exists demon-strating reversal of established effects of digoxin (3-5)
aswellasouabain (6, 7) by specific antibodies or their Fab fragments, and Fab fragments of digoxin-specific
antibodies have recently been used clinically to re-verse toxicity after suicidal digoxin ingestion with
grade atrioventricular block and intractable hyper-kalemia (8).
Thecardiacglycoside digitoxinisused in16-20% of digitalis-treated patients in the United States (9, 10) and is in even more common use in several other countries (2, 11, 12). Substantial differences exist in
plasma protein binding and pharmacokinetics of digi-toxincompared with shorter-acting glycosides (13,14).
The apolar nature of the digitoxin molecule results in
ahigh degree of binding to serum albumin and pre-sumably accounts as well for substantially higher
myo-cardial tissue to medium glycoside concentrationratios
compared with digoxin or ouabain in in vitro studies (15, 16). Qualitative as well as quantitative differences inmyocardial binding of digitoxin compared withmore
polar glycosideshavebeen documented byDutta etal.
(17). Potentially important pharmacodynamic differ-ences, including differences in neurally mediated ef-fects, have been observed inrecentstudies (18, 19).
These differences inpharmacokineticsand pharma-codynamics of digitoxinraiseseriousquestions regard-ing the extent to which prior studies of antibody re-versal of digoxin toxicity can be extrapolated to digi-toxin. Therefore, in the present study we have produced and characterized antibodieswithhigh affinity for digitoxin and have examined in a canine
experi-mental model the efficacy of Fab fragments of these antibodies in the reversal of advanced, potentially lethal digitoxin toxicity.
Because the dog, like other common subprimate laboratory animals, displays a pattern of digitoxin phar-macokinetics profoundly different from man (20), we de-termined that the rhesus monkey (Macaca mulatta)
is asuitable experimental model for digitoxin pharma-cokinetic studies inman. Wethenused this speciesto
investigate the effects on digitoxin pharmacokinetics of these Fab fragments and the antibody population from which theywerederived, and totestthe
hypothe-sis that excretion kinetics of a slowly excreted molecule could be enhanced by the administration ofspecific Fab fragments.
METHODS
Antibody production andcharacterization. Digoxin was
conjugated to bovine serum albumin (BSA)' by periodate oxidation and Schiff'sbase formation and reduction (21, 22)
aspreviously described (23). Sheepwereimmunized with the
BSA-digoxin conjugate in complete Freund's adjuvant and
serially boosted and bled (4). Preliminary studies identified
ananimalthatrespondedto immunization with ahightiter of
antibodies that crossreacted strongly with digitoxin, and
pooledantiserumfromconsecutive bleedings of this animal wasused in subsequent experiments.
The IgG fraction was isolated by ammonium sulfate
precipitation (24), and Fab fragments were prepared by 'Abbreviations used in this paper: BSA, bovine serum albumin;PBS,phosphate-buffered saline.
papain digestion as describedby Nisonoff (25). Undigested
IgG was removed by gel filtration chromatography on
Sephadex G-150 (Pharmacia Fine Chemicals, Inc., Piscata-way, N.J.) equilibrated with 150 mMNaCl, 10 mM Na phos-phate, pH 7.4 (phosphate-buffered saline [PBS]). Sodium dodecyl sulfate polyacrylamide-gel electrophoresis per-formed by the method of Weber andOsborn (26),butomitting
the2-mercaptoethanol step,confirmed the absence of
detect-able IgGin pooled Fabpeaks. Digitoxin-binding capacities of FabfragmentandIgG preparations were determinedby a
dextran-coated charcoal method for separation of
antibody-bound and free hapten as previously reported (23, 27).
All antibody preparations were centrifuged at 20,000 g for 20min andpassed throughasterile 0.22 ,um Millipore filter
(Millipore Corp., Bedford, Mass.) just before use. Control
(nonspecific) IgG and Fab fractions were identically
pre-pared from sera of sheep not previously immunized with
cardiac glycosideconjugates.
Associationand dissociationrate constants for interactions
between antibody and digitoxin were determined as
pre-viously described (28, 29). The average intrinsic association constant (K0)of the antibody population studied was deter-mined by equilibrium dialysis as described in detail
else-where (23), using [3H]digitoxin of specific activity 20 Ci/ mmol(New England Nuclear, Boston, Mass.).
Toxicity reversal experiments. 16 mongrel dogs (mean
weight 13.3+1.2 [SEM] kg) were anesthetized with i.v.
pentobarbital (30 mg/kg) and ventilated with a Harvard respirator at 12cycle/minwith a tidal volume adjusted to the
weightofthe animal. Arterial Po2wasmaintained in the range
of95-100mmHg, and electrocardiogramswere continuously
recorded. Preliminarydose-response experiments were
car-riedout to determine a digitoxin dose that would elicit
re-producible endpoints of advancedtoxicity. For the studies
reported here, 0.5 mg of digitoxin/kg body weight was
in-jected intravenously over 10 min. When ventricular
tachy-cardia had ensued for 5 min, eight control dogs were
thengivennonspecific (control) Fab fragmentsintravenously.
Asecondgroupofeight dogs received an amount of specific
Fabfragmentsrepresentingthemolarequivalent of the
digi-toxindoseover 3min,followed bya30-mininfusionof
one-third of the initial dose. Assuming a molecular weight of
50,000 for Fab fragments, the total dose of specific Fab frag-mentsgiven was 44 mg/kg. Blood samples for determination
ofplasma digitoxin concentrations weredrawn at the onset
ofventricular tachycardia and at hourly intervals after ad-ministration of Fab fragments. After 3 h in stable sinus
rhythm, surviving dogs were allowed to breathe
spon-taneouslyand toawaken.Electrocardiograms were again
re-corded24hafter digitoxin administration.
Pharmacokinetic studies. Three male rhesus monkeys
weighing4.6, 7.4, and 13.6 kg were sedated 1 h before
digi-toxin administration with ketamine, 20 mg/kg, to facilitate
handling. This set of experiments comprised four parts.
Initially, animals received 0.2 mg digitoxin in saline
intra-venouslyover 5 min. In the subsequent three phases, each
separated in time by 4-6 wk, the same dose of digitoxin
included 33 ,uCi [3H]digitoxin (sp act 20.0 Ci/mmol, New
England Nuclear) to permit determination of plasma and urinedigitoxinormetabolite concentrations by direct count-ing of radioactivityas well as by radioimmunoassay. Blood
samples in all experiments were obtained at 0.5, 1, 2, 3, 4, 6, 8, 10, 12, 24, 28, 48, 52, 72, 96, and 100 h after digitoxin
administration and also on the 7th, 9th, 11th, 14th, and
21stdays.
Thefourexperimental phases for each animal were as fol-lows: (a)Digitoxin administration followedbycontrol
followed byacalculated dose of 26.2 mg ofspecific Fab
frag-ments 6hlater(2 mol ofbindingsitesper mole ofdigitoxin administered);(c) Digitoxin administration followedbya
cal-culated dose of 39.3 mg ofspecific IgG 6 h later (2 mol of
binding sites per mole ofdigitoxin administered); and (d)
Digitoxin administration without subsequent IgGorFab in-fusion forcomparison with phaseaabove.
No untoward effects of infusion ofsheep IgG or Fab frag-ments were noted.
In the second and thirdphases, additional blood samples
were drawn 6.5,7, 9, and 11 h afterdigitoxin administration (0.5, 1, 3, and 5 hafter specific FaborIgG injections,
respec-tively). Spontaneously voided urine was collected during
time intervals between0and6, 6and 12, 12and24,24and
48, 48 and 72, and 72 and 96 h after digitoxin
administra-tion. All plasma and urine samples were stored at -20°C untilanalyzed.
Plasma and urine digitoxinmeasurements. Digitoxin con-centrations inplasmasamplesfromdogsandinserumsamples
from rhesus monkeys were determinedby radioimmunoassay
aspreviously described(30). Identicalresultswereobtained
forsamples preparedasplasmaor serumfromeither species, and aretherefore reportedasplasmavalues for convenience. Samples obtained afteradministrationofspecificantibodyor Fabfragmentswere diluted1:5 with PBS, heatedfor1 h in a waterbath at 100°C, and centrifuged for 30 min at 5,000 g to removecoagulatedprotein. Suitable amounts ofthe clear supernatant phase were then used to determine the total
digitoxincontent.Initial studies showed 95-98% recoveryof knownamounts of digitoxin by this method whether or not an excess of specific antibodyorFabfragments was present
before theheatingstep. The results tobereported havenot been correctedfor these minor losses.
To determine the effect of[3H]digitoxin present in some doses administered to monkeys on the radioimmunoassay
determination of unlabeleddigitoxin,standards consisting of
aliquots ofsuitable dilutions of the administered digitoxin
mixture were used as well as conventional unlabeled ards. Comparison of results using these two types of stand-ards documented the absence of measurable interference intheradioimmunoassay systembytherelatively low specific activityof[3H]digitoxin administeredtoanimals.
For the direct determination of 3H counts in samples, 0.2 mlof urine or plasma was counted in 10 ml of the liquid
scintil-lation medium describedby Bray (31). Quenching corrections werebased oninternal standards.
Separationof antibody-boundorFabfragment-bound from free and albumin-bound digitoxin in plasma and urine
samples was accomplished by equilibrium dialysis (23).
Dialyses were carried out in plastic chambers (Technilab
Instruments, Inc.,Pequannock, N. J.) with 1-ml volumes on either side of a sheet ofwashed dialysis tubing (average pore diameter 48A,Arthur H.Thomas Co., Philadelphia, Pa.). Plasmasamplesweredialyzedagainst equal volumes of nor-malrhesus or dog plasma, as appropriate, at 4°C with gentle mixingforaperiod of 6 days. Preliminary studies were car-ried out to ensure thatequilibration had fully occurred by this time. Tritium counts on each side of the dialysis
membrane weredetermined by liquidscintillation counting asdescribed above. 1-mlurinesamples were dialyzed against PBSusingthe method justdescribed.
Sincethedigitoxinradioimmunoassayas usedwaslimited
to a sensitivity of about 1 ng/ml, 1-ml samples ofplasma
obtained from dogs 1-6 h after specific Fab fragment ad-ministration were alsoequilibratedfor 4 h with 2 ng of
[3H]-digitoxin added in vitro and then dialyzed against 1 ml of normal dog plasma. This extended the sensitivity of
detec-tion of freedigitoxintolevels ofatleast0.05ng/ml.
Analyticalstudies of digitoxin and metabolites excreted in
urine. Urine samples from rhesus monkeys were heated at 100°C for 1 h in a boiling water bath to denature
ex-cretedimmunoglobulinsorimmunoglobulin fragments. 1ml of urine was then added to 3 ml of dichloromethane (Aldrich
Chemical Co., Inc., Milwaukee, Wis.) and mixed for5 min
with a Vortex-Genie (Scientific Industries, Inc., Bohemia,
N. Y.) in a glass-stoppered tube (32). After centrifugation, aliquots ofaqueous and dichloromethane phases were sub-jected to liquid scintillation counting as described above.
After extraction, another aliquot of the dichloromethane
phase was evaporated in a water bath at 50°C to dryness and the residue redissolved as describedbyStorstein(33). Appro-priate amountsofthe tritiated digitoxin solution administered to animals in the second, third, and fourth experimental
phases and [3H]digoxin (New England Nuclear) were
identicallytreatedand used asreference standards, appliedin
parallel with aliquots ofdichloromethane extracts to
silica-gelchromatographicsheets (EastmanChromagram,Eastman Kodak Co., Rochester, N. Y.). The sheets were run in cyclo-hexane:glacial acetic acid:chloroform, 49:2:49, developed with chloramine-T followed by heating, and read under ultraviolet light (34). Tracks along which applied materials were chromatographed were then cut into 1-cm-long sec-tions and subjected toliquid scintillation counting as noted above.
Pharmacokinetic analysis. Plasma pharmacokinetic data
were fitted by computer using weighted nonlinear-squares
regressionanalysis to afunction of the form
C =Aet-c +Be-"t
where C= plasma concentration and t= timeafter the dose (35, 36). The coefficients A, B, a, and 8 are "hybrid"
quantities related to parameters of a two-compartment open
model(37, 38). Each residualerror wasweighted byafactor equal tothe reciprocal ofthe concentration. Goodness of fit was assessed by comparison of actual data points to the
com-puter-generated line todetermine randomness of scatter. All
analyses werethen repeated using triexponential functions consistent with athree-compartmentopen model. The most appropriate model(biexponentialfor the datareported here)
was chosenaccording to which yielded the smallest sum of squares of residual errors. The following pharmacokinetic
parameters were then calculated: distribution half-life
(tb), apparent elimination half-life
(tip),
volume of centralcompartment(V,),total apparentvolumeof distributionusing the "area" method (Vd), and total clearance.
RESULTS
Characterizationofantibodies withhigh
affinity
fordigitoxin. A sheep immunized and repeatedly boosted with a digoxin-bovine serum albumin (BSA) conjugate produced antibodies that crossreacted strongly with digitoxin. The average intrinsic
associa-tion constant (K0) for digitoxin of the pooled
anti-serum used in experiments reported in this paper, as determined by equilibrium dialysis and Scatchard
analysis, was 1.4 x 1010 M-1 (Fig. 1). Association and dissociation rate constants forthe reaction
kf
antibody +digitoxin antibody-digitoxin complex kr
F 16
12-
8-4
0 - 2 3 4 5
B(x109M)
FIGURE 1 Scatchard plot of data obtained from equilibrium
dialysis of [3H]digitoxinagainstspecificantibody.B denotes
the molarconcentrationof digitoxin boundtoantibody; F is the molar concentration offree digitoxin. The average intrinsic associationconstant(K0)of the antibodypopulationfor digi-toxin is 1.4 x 1010M-l.
The resulting kf:kr ratio of 1.9x 1010 M-l is in
satis-factory agreement with theK0 value of1.4 x 1010 M-l
obtained under equilibrium conditions.
Digitoxin toxicity reversal studies in dogs.
Pre-liminary studies demonstrated thati.v.digitoxin doses between0.15and0.3mg/kggivenover 10 minusually caused ventricular tachycardia within 1 h, lasting 30-90 min. Both time of onset and duration of arrhythmias werevariable, however, and the dose of0.5mg/kg was chosen forsubsequent studies because of the reproduc-ible and unequivocal endpoint of death in all eight
dogs receiving this dose (Table I) without specific Fab infusion.
The usual pattern of digitoxin intoxication was an
TABLE I
Digitoxin Toxicity Reversalin the Dog
Control
(non-specific) Fab Specific Fab
n=8 n=8
Time toVT,* min 23.4±3.8 28.1±2.8 Incidence of death 8 0 Timetodeath, min 101±36
Time toreappearanceof conducted
sinusbeats,min 18±4 Time tostablenormal sinus
rhythm,min - 53.9±16.5 Allvaluesaregiven as mean±SEM.
*VT=ventriculartachyeardia.
initialsinusbradycardia withvarying degrees of atrio-ventricular block shortly after digitoxin administra-tion, sometimes followed by runs of supraventricular
tachycardia. Ventricular tachycardia ensued shortly afterthe appearance of the first premature ventricular depolarizations, at an average time of 23.4+±3.8 (SEM) min after digitoxin injection. Ventricular fibrillation occurred terminally in six of eight control animals;
ventricular standstill occurred in the othertwo. Aver-age survival time of the eight control dogs was 101.4 ±36.1 min afterdigitoxin injection (Table I).
The group of eight dogs subsequently treated with specific Fab fragments developed ventricular tachy-cardiaanaverage of 28±3min afterdigitoxin adminis-tration, similar to the time course oftoxicity in
con-trol animals (Table I). In marked contrast to the con-trolgroup,however,conductedsinusbeatsreappeared inallanimalsgivenspecific Fabfragments,an average
of 18+4 min after initial Fab administration. Stable
sinus rhythm without ventricular or supraventricular
ectopic activity was present an average of 54±16 min after Fab injection (Table I).
Animals treated with specific Fab fragments were
observedovera24-hperiod after theacutephaseof the
experiment, and all appeared healthy.
Electrocardio-2800
-k2400
5
c
z 2
t22000
z U z
0 '- 1600
0
0 a 1200
4
4
0i5
I-
1-IL
eur,,
400J
ONSET OF 1 HOUR AFTER
VT Fab INFUSION
FIGURE 2 Plasma total digitoxin concentrations in individual
2400-2200.
c2000
z
2 18
:i
z 1600
Z 1400-0
z
z 1200 0
a 1000.
X 800.
X 600
0 2
TIME(hours)
FIGURE 3 Time courseofplasma totaldigitoxin concentra-tionsof eightdogs treated with specific Fabfragments(upper
curve);brackets denote1SEabove and below themean.The lower curve shows theexpected declineinplasmadigitoxin
levels in ananimal thatreceived nonspecific Fabfragments
and died220minafter digitoxin administration.
grams recorded 24 h after digitoxin administration showed normal sinus rhythmin allinstances.
Fig. 2 shows the effect of specific Fab fragments
on plasma total digitoxin concentration. Each ofthe
eight animals sotreated showeda substantialincrease
in plasma total digitoxin concentration between the
time of onset of ventricular tachycardia (just before Fab infusion) and 1 h after Fab infusion. The mean
value of762±68 ng/ml at onset of ventricular
tachy-cardia was similarto that of832±56 for control dogs, and increasedto2,143±144 ng/mlat 1 h (P<0.0001).
As illustrated in Fig. 3,peak plasma total digitoxin
concentrations were present 1 hafter initial Fab
infu-sionand remainedatthesehigh levelsover the ensuing 2h.Theonly control animal surviving beyond 1 hafter
onset of ventricular tachycardia showed the expected decline inplasma concentration from 1,000 to 600 ng/
mlat 3h(Fig. 3).Despite these striking rises in plasma total digitoxin concentrations, free digitoxin levels in
samples obtained from 1 to 6 hafter specific Fab
frag-mentadministration were less than 1 ng/ml as deter-mined by radioimmunoassay and were less than 0.1
nglml by direct measurement of dialyzable
[3H]digi-toxin added in vitro.
Digitoxin pharmacokinetic studies. The rhesus monkey was studied with the hope that the digitoxin eliminationrate inthis species would be similar to that inman. This proved to be the case. As shown in Fig. 4, semilogarithmic plots of digitoxin plasma concentra-tion vs. time showed a biexponential disappearance pattern. The mean distribution half-life (tb) ofthe three animals studied on two occasions by radioimmunoassay (phase1and phase 4) was 0.59 h (Table II). The apparent elimination half-life
(ti)
was 135.5 h in the absence of specific antibody or Fab fragment infusion. Infusion of nonspecific (control) IgG or Fab fragments produced no201
i0
-?
I
-4
NI
Is0Q
%4
a%.
0 48 96 144 192 240 0
0
0
.
48 96 144 192 240
HOURS
FIGURE4 Time courseofmeanserumdigitoxinconcentrationsafteri.v.injection of0.2mg
digi-toxin.Values fromradioimmunoassayareshowninthe leftpanel(opencircles)forsixstudiesin
three rhesusmonkeys.Theright panel(solidcircles)showsdataobtainedbydirecttritium count-ingafter administration of[3H]digitoxintothe samethree animals. The lines of best fit illustrated werederivedby computerasdescribedinthetext.
AntibodyModification ofDigitoxin Toxicity and Pharmacokinetics
IOOr
50
z
0
44
I--z
w
0
z 0
oc
U_
E
O C
p
Ca
TABLE II
DigitoxinPharmacokinetics in the Rhesus Monkey*
Radioimmunoassay Tritium counts Mean(+SEM) Mean(+SEM)
(n=6) (n=3)
tb, h 0.59(±0.23) 0.86(±0.05)
tp,h 135.5(±+21.9) 117.6(±+10.9)
VI,literlkg 0.20(±0.11) 0.26(±0.03)
Vd,literlkg 1.23(±0.05) 1.26(±0.12) Clearance
mllmin 0.90(±0.22) 0.79 (±0.36)
ml/min per kg 0.113(±0.024) 0.126(±0.017)
VI=volume of central compartment; Vd= volumeofdistribution.
*Data analyzed on the basis of a two-com]
model.
discernible changeindigitoxinpharmaco
pared with experimental phase 4, in wh
alone was administered.
Samples analyzed by directtritium cou results similar to thoseobtainedby radio:
(Table II). Fig. 4 illustrates the good aM tween pharmacokinetic data derived fro pearance of radioactivity from plasma v
tained by radioimmunoassay.
Effects of specific Fab fragments onser concentrations. Asshown in Fig. 5, adr 2molofFabfragments per mole of digito
digitoxin infusion ledtoarapid4.3-foldin(
plasma radioactivity, compared with co
Plasma total digitoxin concentrations tI
25r-loo1
75
50_
25_
E.k
I!@
0 t 24 48 72
HOURS
TIME AFTER DIGITOXIN
FIGURE 5 Effect ofi.v. administrationofspe ments on serumtotaldigitoxinconcentrations
bindingsitespermole ofadministered digitox
hafterani.v.dose of0.2mgdigitoxin. Solidcirc
values for controlexperimentsinthree rhesusn
circles show similarmeanvalues until administ
6h (arrow). Notebreak intime scaleafter96
total apparent partment open
kinetics
com-iich digitoxin
ntingyielded
immunoassay z
a
0
_ 200
z
x c
o~ 100
> 50
en
-5 LUE
"I-. ~
'Ir-
F-ii.
.c1\
r
"I
_I
-?
4
I
N".
° T 24 48 72
HOURS
TIME AFTER DIGITOXIN
96 7 9 11 14
DAYS DOSE
FIGuRE 6 Effect of i.v. administration of specific IgG on
serumtotaldigitoxinconcentrations. 2 mol ofantibody bind-ing sites per mole ofdigitoxin was given 6 h (arrow) afteran i.v. dose of0.2mg digitoxin.Values shown are means from studies of three rhesus monkeys as shown in Fig. 2. Solid circles: control experiments; open circles: specific IgG administration at6h. Note break intime scale after 96h.
greement be- withaninitialmeanhalf-life of about 4 h. 12-16 h after
im the disap- specific Fab injection, plasma concentrations of digi-vith data ob- toxin returned to levels found in control experiments
in which no
specific
Fabfragments
were given.rumdigitoxin Effects ofspecificIgGadministrationonplasma dig-iinistrationof itoxin concentrations. Administration of 2 mol of
spe-oxin 6h after cific binding sitespermole of digitoxin in the form of creaseintotal intact IgG resulted in a substantial increase in total
)ntrol values. plasma radioactivity to a mean peak value 12.9-fold ien declined above pre-IgG infusion levels at the 28th hour of the experiment,asshowninFig. 6. Thiswasfollowed bya
gradual decline of plasma radioactivity until the 4th
day, after which a more rapid fall was observed be-tweenthe 4th and 9th days after specific IgG
admin-istration. Plasma levels of radioactivity were 10-fold
higher 6 and 12 h after specific IgG administration when compared with the last sample obtained before antibody infusion. 2 wk after specific IgG
administra-tion, meanplasma radioactivity concentrations had
re-turned to levels comparable with those observed in control experiments.
Effects of specific Fabfragments on urinary excre-tion ofdigitoxin. Mean urinary excretion of
radioac-tivity over 96h inthe absence of specific antibody or
t7 9 11 14 Fabfragments totaled 11.7±2.4 ug, or 6% ofthedose DAYS given, the largest amounts being
excreted
during theDOSE first and second
6-h
collection periods (Fig. 7).zcific Fabfrag- After infusion ofspecific Fab fragments, urinary
ex-;.2mol of Fab cretion of radioactivity increased 13-fold in comparison
cinwasgiven6 to the first
6-h
period of the experiment (before Fabens
kysh
Opean
administration)
and 12-fold(from
165+61to1,985±395
'ration of Fab at ng/kg, P < 0.05)overrecovery values for thesametime
h. period incontrol experiments in which no Fab or IgG z
0
o^
z
w
u
z
xc
0
a
a)
2r%
CONTROL Fob
HOURS AFTER DIGITOXIN DOSE
FIGURE 7 Cumulative urinary excretion ofdigitoxin and metabolites, determined fromtritiumcountsexcreted after i.v.
administration of 0.2 mg [3H]digitoxin. Solid circles: mean
values for control experimentsinthree rhesusmonkeys;open
triangles:meanvalues forthesamethree animalsgivena
two-fold molar excess of specific Fab fragments at6h (arrow);
opencircles:meanvalues for thesamethree animalsgivena
twofold molar excess of specific bindingsites as intactIgG
at6h.
wasgiven. Tritium recoveryin urineincreased froma
control value of 0.6% to 8% of the [3H]digitoxin dose givenfor the 6-htimeperiodafter specific Fab infusion.
Fig. 8 summarizes the urinary excretion of digitoxin and metabolites during the 6-h collection period
im-mediately after specific Fab administration.Themean hourlyexcretion rateofradioactivity during the 12-to
24-h collection period (6-18 h after Fab infusion)
re-mainedelevated and exceeded values before Fab
ad-ministrationbyafactor of2.2(mean489ng/hvs.220ng/ h). Cumulativeurinary excretion of3Hcountsoverthe
90 h after specific Fab infusion more than doubled
whencomparedwith controlexperiments, asshownin Fig. 7.
Effects of specific IgG onurinaryexcretion of
digi-toxin. In markedcontrast toFabinfusion,
administra-tion ofintactantibodies significantlyreduced the
uri-nary digitoxin excretion rate to 37% of control during the interval between the6th and 12thhourofthe
exper-iment.These dataaresummarizedinFigs.7and 8.
Dur-ing the 90 hafter IgGinfusion, only 1.5% of 3Hcounts presentinthe digitoxindose givenwasrecoveredinthe
urine,compared with 4.1%incontrolexperimentsand
11.4%after specific Fab administration.
Binding of digitoxininplasma andurinebyspecific
IgG and Fab. Table III summarizes results of
equi-librium dialysis studies of plasma samples obtained
after injection of specific IgG or Fab fragments.
FIGURE 8 Mean urinary excretion values for digitoxin and metabolites during the period from 6 to 12 h after i.v.
admini-stration of0.2 mg [3H]digitoxin in three rhesus monkeys. Bracketsshow1SEM. Values given were determinedby
tri-tium countsexcretedin controlexperimentsandafterspecific
Fab or IgG, given at 6 h.
Whereasdigitoxin fullyequilibratedacrossthe dialysis membrane in samples obtained before specific
anti-body orFab injection or after nonspecific protein ad-ministration, the glycoside was morethan 90% bound tospecificIgG orFabinsamples obtained from30min through 5 hafter specificFab or IgG was given. Whereas the amount ofdigitoxin bound to IgG declined insig-nificantly from 99.6 to 98.7% over 90 h, the per-centage ofplasma[3H]digitoxinboundtoFab declined relatively rapidly after 2 h and only a minor fraction
TABLE III
Equilibrium Dialysis of Rhesus Monkey Plasma Samples Obtained afterSpecificFab or IgGAdministration*
Percentbound Percent bound Hoursafterinjection after Fab after IgG ofspecificFaborIgC Mean(±SEM) Mean(±SEM)
h % 3
0.5 99.0(±0.1) 99.6(+0.1)
1.0 98.6 (+0.2) 99.7(+0.03)
2.0 97.2(±1.0) 99.6(±0.1)
3.0 93.5(±5.2) 99.6(±0.1)
4.0 94.3(±3.8) 99.6(+0.04)
5.0 90.6 (+4.1) 99.6(±0.1)
6.0 82.8(±5.9) 99.5(±0.2) 18.0 62.6 (±1.9) 99.8(±0.1) 42.0 12.7 (±1.6) 99.6(±0.1)
66.0 1.6(±0.9) 99.6(±0.04)
90.0 0.5(±0.4) 99.7(±0.4) *Nobindingother than that attributed to serum albumin was
demonstrable in plasma samples obtained before antibody infusion.
z
c;0
P
w
Ir 2(
0) x
w 4-..
D I!(
w
-j
o
^
Z
20F-0'
z 0
w
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w
I-cz 0
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15'-
1o0-5
IgG ----A
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TABLE IV
Equilibrium Dialysisof RhesusMonkey UrineSamples
Ob-tained after Administration of Specific FaborIgG*
Percentbound Percentbound after Fab afterIgG
Collection period Mean(+SEM) Mean(±SEM)
h % %
0-6 2.7(+1.6) 0.3(+0.3)
6-12 98.7(+0.5) 45.2(±12.1)
12-24 77.7(±7.9) 25.3(±9.7)
24-48 58.5(±9.7) 42.0(±8.4)
48-72 40.5(±1.8) 40.0(±5.4)
72-96 17.7(±5.6) 45.6(+3.8)
*No binding was demonstrable in urine samples obtained before antibody infusion.
(12.7%)was stillbound42hafterspecific Fab injection, decreasing to anaverage of1.6%after66h.
The urine equilibrium dialysis results (Table IV)
show amarked increase of the bound (nondialyzable)
fraction of digitoxin duringthe 6 h after specific Fab administrationto98.7±0.5%,comparedwitha
negligi-ble valueof2.7±1.6%forurineexcretedduringthe 6-h
controlperiodbefore Fab infusion. After specific IgG
infusion, the small amount of 3H activity excreted
in-creased from negligible percent bound values during
the control (0-6 h) period to values in the 25-45%
range,presumably relatedtosmallamountsof
immuno-globulin or immunoimmuno-globulin fragments excreted.
Itshould be noted that the percent bound values for urine samples after Fab or IgG administration repre-sent lower limits of actual binding since some degree ofdegradation ofantibodyor antibody fragments dur-ing collection and storageperiodscannotbeexcluded. Analytical studies of digitoxin and metabolites
ex-creted in urine. To testthe hypothesisthat
enhance-mentof3Hexcretion in urineafterspecific Fab infusion
was dueto excretion ofnative digitoxin boundto Fab fragments, further analytical studies were done.
Di-chloromethane extraction according to the method usedshowed that99%of3Hcountsindigitoxin samples before administration to animals were extracted into
the dichloromethane phase. Table V summarizes the percentages of dichloromethane-extractable radioac-tivity from urine obtained during the control
experi-ments (phase 4) and after administration of specific Fabfragments and specific IgG (phases2and 3). After
administration of specific Fab fragments, the percent-age of extractable radioactivityincreasedfromacontrol meanof40%(phase 4) to 92%during the first 6-h pe-riod, gradually decreasingtocontrol levelsbythe time of the 72- to 96-h urinesample. This isconsistentwith accelerated excretion ofnative digitoxin and excludes thepossibilitythat 3Hexcretion was dueto3Hexchange
Smith
with water-soluble material or accelerated formation of water-soluble metabolites. Analysis of extracted
mate-rial bythin-layer chromatography further demonstrated identical mobility of greater than 90% of3H counts pres-ent inthis fraction with identically treated samples of [3H]digitoxin standard.
DISCUSSION
Digitoxin is one of the two cardiac glycosides most widely usedin clinical practice. A substantial number of instances ofsevere intoxicationhave been reported (2, 39). Due to its prolonged half-life compared with other glycosides (13), digitoxin intoxication poses a special therapeutic problem.Inaseriesof 115 patients treatedfor advanced (usually suicidal) digitalis intoxi-cation,96%had taken digitoxin and the resulting
mor-tality was 22%(2). Patients died as late as4 days after injection of the drug, and doses as low as 3 mg were reported to cause mortality. Caldwell et al. have sug-gested a novel therapeutic approach using oral ingestionofasteroid-bindingresintointerrupt entero-hepaticcycling of digitoxin (40, 41). This approach was highlyeffective in the rat (40), a species with very
ac-tive enterohepatic cycling ofdigitoxin, and some
en-hancement of digitoxin excretion was also documented in man (41). However, since no specific antagonist of demonstrated clinical effectivenessinthesetting of ad-vancedtoxicity has yet been described, therapy is re-stricted to symptomatic management of the clinical manifestations of digitalis toxicity.
To provide a more specific and effective therapeutic approach, the present study was undertaken to deter-minewhether specific Fab fragments of antibodies that bind digitoxin with high affinitycan reverseadvanced, life-threatening digitoxin toxicity in a canine experi-mental model. The feasibility of this approach was suggested by earlier studies using digitalis-specific antibodies ortheir Fab fragments for reversal of
glyco-TABLE V
Percentof3HRadioactivity Extractableinto
Dichloro-methanefrom Rhesus MonkeyUrine Samples*
Phase2 Phase3 Phase4
Collection (specific Fab) (specific IgG) (noFaborIgG)
period Mean(+SEM) Mean(tSEM) Mean(tSEM)
h
0-6 38.2(±2.2) 37.8(±5.1) 40.0(±2.1)
6-12 91.8(+2.4) 37.7(±5.3) 49.7(+0.2)
side-induced effectsin vitro(4-7, 42-44)and toxic
ar-rhythmias in vivo (3-5,45). Differencesinboth pharmaco-kinetics (13, 46) and pharmacodynamics (15-19),
how-ever,makeextrapolation of results fromstudies of one
cardiacglycoside to another hazardous. The rationale foruseof Fabfragmentsinpreferenceto intactIgGfor
in vivo reversal of cardiac glycoside toxicityhas been
discussedindetail recently (5, 47) and includes lesser
antigenicityof FabcomparedwithIgGas well as more
rapid excretion of both binding protein and bound drug.
Withregard to potential therapeutic use, thefirst req-uisite of an antibody population or the Fab fragments derived therefromishigh affinityandspecificityfor the
drug molecule whichisto be counteracted. In thecase
of cardiac glycosides such as digitoxin and digoxin, toxic effects may be manifestatfreeplasma concentra-tions of10 nM or less (30, 48). Antibody affinity con-stants musttherefore beatleast109 ormoreif free gly-coside concentrations are to be lowered to negligible levels without the use ofexcessive amounts of
anti-body, an issue of obvious importance ifheterologous
antiserum is used as the source of cardiac
glycoside-binding immunoglobulin (5).
Inextendingourearlier worktothe reversal of digi-toxin toxicity,wehave takenadvantage of the
observa-tion that selected antisera from animals immunized
withBSA-digoxin conjugates containantibody
popula-tions that strongly crossreact with digitoxin (23). Among the firstthreesheep screened, weidentifiedananimal thatdevelopedahightiterof antibodieswith aKo value fordigitoxin of1.4 x 1010 M-1by equilibrium dialysis,
in satisfactory agreementwith results of separate esti-mates of the kinetics of formation and dissociation of the hapten-antibody complex. The rapid second-order association kinetics ensure that the free fraction of serumdigitoxin concentration will falltoless than1 nM
within afew seconds of the infusion of antibodyinthe
amountusedinthe experimentsreported here,
assum-ing rapid mixing in the intravascular compartment. This rapid binding of digitoxin by antibodyoccurs in
spite ofextensive serum albuminbinding ofdigitoxin
(14) because of the very rapid dissociation rate of the digitoxin-albumin complex. Itshouldbenoted that the degree of digitoxin crossreactivity noted in the present studies cannotbe expected for every digoxin-binding antibody population (23).
The dose of0.5mg/kgofdigitoxin administered
intra-venouslytodogs uniformly led tothe deathof the ani-malsat amean timeof101min.Nonspecific Fab given
to eight control animals did not protect from digitoxin toxicity. The administration of a small (33%) molar
ex-cess of specific Fab fragments not only prevented the
deathofalleightanimals sotreated, butalso reversed
cardiotoxiceffects completelyandled toreappearance of conducted sinus beats as early as 9-10 min after
injection. This relatively rapid reversal of established advanced digitoxin toxicity confirms and extends pre-vious experience with antibody reversal of established digoxin toxicity (3-5) as well as our initial clinical experience in the treatment of advanced digoxin tox-icity with purified specific Fab fragments (8).
Arapid increase inplasma total digitoxin levels took place within 30 min after the end of specific Fab infu-sion in all dogs (Fig. 2); during the subsequent 2 h, plasma concentrations did not change significantly (Fig. 3). A similar plateau of serum total digoxin con-centration after the initial rise after administration of specific Fab fragments lasted for 10 h in a patient treated for advanced digoxin toxicity after a massive su-icidal ingestion of 22.5 mg of the drug (8). The
consis-tentincreaseintotal digitoxin concentration inplasma observed after specific Fab administration in the stud-ies reported here presumably reflects continuing re-moval of digitoxin from the tissues and sequestration in the extracellular space in an antibody-bound, phar-macologically inactive form, balanced by renal
excre-tionof the Fabfragment-digitoxin complex. This
mech-anism was further supported by equilibrium dialysis studies showing free digitoxin concentrations of less than0.1
nglml
after specific Fab infusioninthese dogs. The prolonged half-life of digitoxin in man is asso-ciated with very slowexcretion bythekidneys, which in turn is presumably due to the high degree of binding ofdigitoxininthecirculationbyserumalbumin. Lukas and DeMartino demonstrated that 97% of digitoxin at usual therapeuticconcentrations isbound toserum al-bumin (14). Aneffectiveapproachtothe specific treat-ment of advanced digitoxin intoxication, then, ideally should enhanceexcretion of the drug as well as neutral-ize its effects before excretion.Canine digitoxin excretion, like that of usual labora-tory animals such asthe mouse, rat, and cat, is substan-tially more rapid thanisthe caseinman (11,13,20). The rhesus monkey (Macaca mulatta) was shown in the present studies to provide a more suitable model. The mean elimination half-times of 135 and 118 h found by
two separatemethods inthis species (Fig. 4; Table II)
are insatisfactory agreementwiththe values of 115 and
165h in manreported by Lukas (13) and Gjerdrum(11),
respectively.
Prior studies ofthe effects of digoxin-specific IgG and Fab fragments on the pharmacokinetics of digoxin in the dog have documented substantial rises in plasma total digoxin concentration, with IgG causing about a sixfold greater rise than Fab (47). Analogous results
were observed in the present rhesus monkey
experi-ments. Administration of 2 mol of binding sites per mole of digitoxin in the form of an IgG
prepara-tion resulted in a greater and substantially more prolonged increase in plasma total digitoxin levels compared with the response to an equal number
of binding sites given as Fab fragments. The
dif-ferences may be accounted for in part by a smaller distribution space of 160,000-dalton IgG molecules
compared with 50,000-dalton Fabfragments, as well as by continuing urinary excretionofFab fragments. The
increases in plasma total digitoxin levelsafterboth IgG
and Fab administrationweredemonstrated in
equilib-riumdialysis studiesto beduetohigh-affinity
immuno-globulin binding, andwere accompanied by rapid
de-creases in free digitoxin concentration to near-zero
levels (Table III).
Administration ofIgGreduced theurinary excretion
of radioactivityto37%ofcontrol values. Of thisamount
morethan40%wasnotdialyzable,indicating that
digi-toxin waspartly excretedinboundform, possiblytolow molecular weight antibody fragments formedin vivo.
The serum
ti
of homologous and heterologousy-glob-ulininjectedintorabbitsorguinea pigs hasbeen shown
to be between 4.2 and 6 days (49), and only small
amountsofintact IgG wereexcretedinthe urine.The reduction in urinary excretion of radioactivity after specificIgGadministration wastherefore notan
unex-pected observation.
Binding of digitoxinto Fabfragments appears to ac-count for the initial half-life of digitoxin elimination
from serum of4 h (Fig. 5). It has been reported that rabbitFabfragmentsareexcreted bythe mouse, at least in part viathe kidneys, with a half-time of3.6 h (50).
Although comparable data are not available for sub-humanprimates,the data of Janewayetal. (51) indicate
elimination of immunoglobulin fragments ofsize and otherproperties similartoFab by humans witha
half-timeof5 h.Onewould therefore predictthatrelatively rapid renal elimination of sheep specific Fab fragments by therhesus monkey kidneywould enhanceexcretion
of bounddigitoxin, andthis proved to be the case, as
summarized in Figs. 7 and8. The high affinity of this population of Fab fragments for digitoxinensuresthat,
oncefilteredbytheglomerulus,theFab-digitoxin
com-plex will notdissociate toamajorextentand the
digi-toxin excreted in the urine remains predominantly bound in early urine samples collected after specific Fab administration (Table IV). The analytical
experi-mentsusingdichloromethaneextractionandthin-layer
chromatography suggest thatadministration ofspecific Fabfragments ledtoelimination ofnativedigitoxin and
notof metabolites.
Under theexperimental conditions usedinthis pri-matemodel,digitoxin elimination rose 14-foldto 8%of
thedosegivenover 6h, compared with controlvalues.
When in severe digitoxin intoxication an even more
pronounced acceleration of glycoside excretion is de-sirable, this may well be achieved byaconstant
infu-sionorrepeateddosesofpurified Fab fragments over a moreprolonged periodoftime. This,toourknowledge,
is the first demonstration of enhanced excretion of a
drug byan immunoglobulin fragment and may repre-sentonly one example ofa number of therapeutic op-portunitieswhereinahigher clearance ofa drug or hor-mone can be obtained.
In conclusion, the present studies demonstrate that high-affinity specific Fab fragments enhance urinary excretionof digitoxin, whereas administration ofintact IgG leads to the opposite effect and retainsthe poten-tially toxicglycosidestores in the body. Taken together
withthetoxicityreversal studies in the dog, these data support the potential clinical use of purified specific Fab fragments in selected cases of advanced,
life-threatening digitoxin toxicityunresponsiveto conven-tional therapy.
ACKNOWLEDGMENTS
This work wassupportedinpart by Award HL-18003 and Pro-gram ProjectAwardHL-19259,both from the National Heart, Lung,and BloodInstitute.
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