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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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(2)

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 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.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

(3)

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

(4)

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 central

compartment(V,),total apparentvolumeof distributionusing the "area" method (Vd), and total clearance.

RESULTS

Characterizationofantibodies withhigh

affinity

for

digitoxin. 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

(5)

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

(6)

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 no

201

i0

-?

I

-4

NI

Is

0Q

%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

C

a

(7)

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

Fab

fragments

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 the

DOSE 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 Fab

ens

kysh

Opean

administration)

and 12-fold

(from

165+61to

1,985±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%

(8)

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

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Ir 2(

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w 4-..

D I!(

w

-j

o

^

Z

20F-0'

z 0

w

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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)

(10)

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

(11)

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 heterologous

y-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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