AJH 1997; 10:332 – 336

BRIEF COMMUNICATIONS

Hemodynamic Effects of the Fab Fragment

of Digoxin Antibody (Digibind) in
Corticotropin (ACTH)-Induced Hypertension
Ming Li, Cheng Wen, and Judith A. Whitworth

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To determine whether the immune Fab fragment digibind decreased MAP (014 { 3 mm Hg, P
of digoxin antibody (digibind) attenuates õ.001) in ACTH hypertensive rats, but not in
established corticotropin (ACTH) hypertension, ACTH sham control normotensive rats (/2 { 3
rats were given ACTH sham control (0.1 mL mm Hg). Blood pressure reached a minimum after
normal saline, twice daily, subcutaneously, n Å 14 { 3 min and the effect lasted more than 30
18) or ACTH treatment (2.5 mg/kg in 0.1 mL min. No significant change of blood pressure was
normal saline, twice daily, subcutaneously; n Å found in ACTH treated rats receiving sham (0.9%
27) for 10 days. Acute hemodynamic effects of NaCl) digibind injection (/2 { 2 mm Hg).
digibind (30 mg/kg, intravenous bolus injection) However, both preimmune sheep IgG and IgG
were examined after 10 days of sham control or (Fab)2 fragment caused a decrease of blood
ACTH treatment. Rats were divided into 7 groups: pressure in both sham or ACTH treated rats.
digibind (30 mg/kg, in 1 mL 0.9% NaCl Although these data that digibind decreases BP in
intravenous bolus injection) plus sham (n Å 6) or ACTH but not sham treated rats are consistent
ACTH (n Å 8), sham digibind (1 mL 0.9% NaCl with the notion that digitalis-like substances may
intravenous bolus) plus ACTH (n Å 7), digibind play a role in ACTH induced hypertension, the
vehicle (sorbitol 1.8 mg in 1 mL 0.9% NaCl, evidence that both preimmune sheep IgG and IgG
intravenous bolus) plus sham (n Å 6), preimmune (Fab)2 fragments also decreased blood pressure in
sheep IgG (30 mg/kg in 1 mL 0.9% NaCl rats suggests caution in interpretation of studies
intravenous bolus) plus sham (n Å 6) or ACTH (n that employ digibind preparations. q 1997
Å 6) and preimmune sheep IgG (Fab)2 fragment American Journal of Hypertension, Ltd. Am
(30 mg/kg in 1 mL 0.9% NaCl intravenous bolus) J Hypertens 1997; 10:332 – 336
plus ACTH (n Å 6). ACTH increased systolic
blood pressure (SBP) from 118 { 2 to 132 { 3 mm KEY WORDS: Adrenocorticotrophin (ACTH), blood
Hg on treatment day 10. BP was unchanged in pressure, digitalis-like substances, digibind,
sham treated rats. The acute administration of hypertension, rat.

D
igitalis-like substances (DLS), which in- body to rats decreased blood pressure in DOCA-salt,3–5
hibit Na, K-ATPase, have been repeatedly 1 kidney, 1 clip,4 and aortic coarctate hypertension,6 but
demonstrated in vascular smooth muscle not in spontaneously hypertensive rats (SHR).4 How-
and myocardium of animals with various ever, Mann et al5 demonstrated that the Fab fragment
forms of ‘‘volume-expanded’’ experimental hyperten- of digoxin antibody (digibind, intravenously) did not
sion.1,2 The acute administration of antidigoxin anti- alter blood pressure in DOCA-salt hypertensive rats. In

Received June 20, 1994. Accepted August 5, 1996. Address correspondence and reprint requests to Judith A. Whit-
From the Department of Medicine, St. George Hospital, Kogarah, worth, Department of Medicine, St. George Hospital, Kogarah,
New South Wales, Australia. NSW 2217, Australia.

q 1997 by the American Journal of Hypertension, Ltd. 0895-7061/97/$17.00

Published by Elsevier Science, Inc. PII S0895-7061( 96 ) 00318-4
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AJH–MARCH 1997–VOL. 10, NO. 3, PART 1 HEMODYNAMICS OF DIGIBIND IN ACTH-HYPERTENSION 333

contrast, a recent study7 reported that digibind de- pressure transducer (World Precision Instruments, Inc.,
creased blood pressure in DOCA-salt hypertensive rats. Sarasota, FL) to pressure monitor BP-1 (World Precision
Corticotropin ( ACTH ) administration reproducibly Instruments, Inc., Sarasota, FL).
increases blood pressure in the rat, and this effect is Digibind, Digibind Vehicle (Sorbitol), Digibind
adrenally dependent.8 ACTH also increases plasma Control [Sheep IgG and Sheep IgG (Fab)2 Fragment]
immunoreactive DLS concentration in the rat, and Administration Digoxin immune Fab (ovine) digi-
there is a significant correlation between plasma DLS bind (The Wellcome Foundation Ltd., Beckenham,
and systolic blood pressure (SBP) (r Å 0.53, P õ.001, Kent, England) was reconstituted with sterile water for
n Å 44).9 Ludens et al10 suggested that the adrenal injection BP (Astra Pharmaceuticals Pty. Ltd., N. Ryde,
cortex may be a major contributor to circulating DLS NSW, Australia) and diluted to desired protein concen-
in the rat. These data led us to propose that DLS may tration (between 9 and 11 mg/mL) with sterile saline
play a role in ACTH-induced hypertension. 9 This (0.9% NaCl) for injection BP (Astra). Sorbitol was dis-
study examined the ability of digibind to antagonize solved in sterile saline for injection BP (1.8 mg/mL).
ACTH-induced hypertension in rats. Sigma’s (Sigma Chemical, St. Louis, MO) purified sheep

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IgG and IgG (Fab)2 are essentially salt-free. They were
METHODS
reconstituted with sterile saline for injection BP (0.9%
Experimental Animals These studies were approved NaCl, 150 mmol/L), thus producing a protein concen-
by the Animal Care and Ethics Committee of the Uni- tration of between 9 and 11 mg/mL.
versity of New South Wales. Forty-five male Sprague- The osmolalities of the solutions were measured us-
Dawley rats (292 to 331 g body weight) were used. Each ing a freezing-point osmometer (Gonotec, Gesellschaft
day at 16:00 rats were offered 60 g of commercial diet für Meß- und Regeltechnik mbH, Berlin, Germany):
(containing 0.5% salt; Y.S. Feeds Pty. Ltd., Sydney, New 0.9% NaCl solution 285, sorbitol solution 297, digibind
South Wales, Australia) and demineralized water ad 290, sheep IgG solution 347, and sheep IgG (Fab)2 frag-
libitum. Twenty-four hour water intake and urine vol- ment solution 334 mOsm/kg.
ume were measured every fourth day in metabolic Fourteen rats received digibind (30 mg/kg in 1 mL,
cages. At least 6 days control measurements were ob- bolus, sham n Å 6; ACTH n Å 8). Twelve sham-
tained. At the end of the experiment rats were killed treated rats were given either sorbitol (1.8 mg/kg in
under nembutal anesthesia and blood samples were 1 mL, intravenous bolus, n Å 6) or a preimmune
taken from the carotid artery. sheep IgG (30 mg/kg in 1 mL, intravenous bolus, n
Å 6) as digibind sham control. Nineteen ACTH-
Experimental Protocol Rats were divided into two treated rats were given 0.9% NaCl (1 mL intravenous
groups: sham and ACTH treatment. Control days (prior bolus, n Å 7), preimmune sheep IgG (30 mg/kg in 1
to any treatment) are designated C, and days of treat- mL, n Å 6) or a preimmune sheep IgG (Fab)2 fragment
ment with sham (saline) and ACTH as T. (30 mg/kg in 1 mL, intravenous bolus, n Å 6) as digi-
Sham Treatment Following 6 control days (C1-6), rats bind controls. Digibind or control injections were
(n Å 18) received 0.9% NaCl (0.25 mL/kg, twice daily, given after 10 days of sham or ACTH treatment and
subcutaneously) at 10:30 and 17:30 as a sham control effects on BP determined. Carotid arteries and jugular
for ACTH injection for 10 days. veins were cannulated under nembutal anesthesia (60
mg/kg, intraperitoneally). Mean arterial blood pres-
ACTH Administration Following 6 control days (C1- sure was monitored for 15 min before and 45 min
6), rats (n Å 27) were given synthetic ACTH (Synacthen after injection of digibind or digibind controls. At
Depot, Ciba-Geigy, Sydney, Australia) 5 mg/kg/day death, adrenal, kidney, and heart weight/body
(2.5 mg/0.25 mL twice daily) subcutaneously at 11:30 weight ratios were measured.
and 17:30 for 10 days. Statistical Analysis Results were expressed as mean
Blood Pressure Measurements Blood pressure was { SEM and analyzed using two-way repeated measure
recorded on alternate days by the tail cuff method9 in analysis of variance (ANOVA) to compare two groups
conscious rats. At death, BP was measured both by the together, and then a single treatment group to the con-
tail cuff method and directly. Direct arterial pressure trol group, and to analyze a single treatment group
was recorded using cannulation of the carotid artery. over time. The pretreatment days were pooled to give
Nembutal anesthesia was maintained during blood a mean pooled control (PC). If these were significant,
pressure recordings. A polyethylene catheter with an an unpaired Student’s t test was performed. The P value
outer diameter of 0.94 mm and inner diameter of 0.75 for the t test was modified by the Hochberg method of
mm (Portex, Boots Pty. Ltd., Melbourne, Australia) was multiple comparisons.
used for cannulation of the left carotid artery via a mid- RESULTS
line neckline incision. The catheter was connected to a Effects of Sham and ACTH Treatments Sham treat-
three-way stopcock that was connected directly by a ment (n Å 18) did not change SBP. There was a signifi-

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334 LI ET AL AJH–MARCH 1997–VOL. 10, NO. 3, PART 1

cant increase in body weight (T9 0 T0 / 45 { 8 g,

P õ.001), but no changes in water intake and urine
volume.
ACTH treatment (n Å 27) increased SBP from 118 {
2 to 132 { 3 mm Hg on treatment day 10. There was a
significant increase in body weight (T9 0 T0 / 40 { 5
g, P õ.001), but no changes in food intake, water intake,
and urine volume.

Digibind, Digibind Vehicle, and Preimmune Sheep

IgG Administration in Sham Treated Rats The acute
administration of digibind or digibind vehicle (sorbitol)
did not influence blood pressure in the sham group.
However, bolus injection of preimmune sheep IgG sig-

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nificantly decreased MAP (030 { 4 mm Hg, P õ.001)
in sham treated rats. Blood pressure reached a mini-
mum after 1 { 0.2 min and the effect lasted more than
35 min.

Digibind, Sham Digibind, Preimmune Sheep IgG,

and Preimmune Sheep IgG (Fab)2 Fragment Adminis-
trations in ACTH Treated Rats Injection of digibind FIGURE 1. Mean arterial pressure (MAP) in sham plus digi-
decreased MAP (014 { 3 mm Hg, P õ.001) in ACTH bind vehicle (n Å 6), ACTH plus sham digibind (saline) (n Å 7),
and ACTH plus IgG (Fab)2 fragment (n Å 6) treated rats. Statis-
treated rats. Blood pressure reached a minimum after
tics compare precontrol (0) with treatment time (min) (*P õ.05;
14 { 3 min and the effect lasted more than 30 min. In **P õ.01; ***P õ.001).
contrast, 0.9% NaCl injection did not alter blood pres-
sure in ACTH treated rats. Preimmune sheep IgG also
significantly decreased MAP (035 { 7 mm Hg, P õ.001)
in ACTH treated rats. Blood pressure reached a mini- Preimmune sheep IgG also decreased blood pressure
mum at 1 { 0.2 min and the effect lasted more than to similar degrees in both sham and ACTH treated rats.
40 min. Preimmune sheep IgG (Fab)2 fragment bolus With preimmune sheep IgG administration blood pres-
injection decreased MAP (0 35 { 6 mm Hg, P õ.001) sures were consistently higher in ACTH treated rats
at 35 { 3 min, maximum at 45 { 5 min. (055 mm Hg, than in sham treated rats (P õ.01 between groups, Fig-
P õ.001) in ACTH treated rats. ure 3).
DISCUSSION
Intergroup Comparisons SBP in ACTH treated rats
was higher than in sham treated rats (T10 sham 115 { Numerous studies have confirmed the presence of
2 v ACTH 132 { 3 mm Hg, P õ.001). At death, direct DLS in blood, urine, cerebrospinal fluid, and various
arterial pressure was also higher in ACTH treated rats tissues including the adrenal, heart, and hypothala-
(MAP 119 { 3 mm Hg, P õ.01) than in sham rats (MAP mus,10,11 and DLS may play an important role in blood
96 { 2 mm Hg). pressure regulation.1 – 5 Acute administration of antidi-
There were no changes of MAP in sham treated rats goxin serum (ADS) caused a pronounced long-lasting
with digibind vehicle (sorbitol) (Figure 1) or digibind blood pressure decrease in hypertensive rats treated
injection (Figure 2), nor in ACTH treated rats with sham with DOCA and 1% saline from prepuberty.3,4,7 The
digibind (0.9% NaCl) administration (Figure 1). In decrease of blood pressure was only moderate in 1K-
ACTH treated rats preimmune sheep IgG (Fab)2 frag- 1C Goldblatt rats, whereas there was no change of
ment administration also significantly decreased blood blood pressure after ADS injection in spontaneously
pressure (P õ.001) compared with ACTH plus sham hypertensive rats.4 ACTH increases plasma DLS con-
digibind (Figure 1), but the time courses were different. centration in the rat, and there is a close correlation
MAP was decreased by digibind at 10 { 2 min, reached between plasma DLS concentrations and SBP.9 In the
a minimum after 14 { 3 min, and the effect lasted 30 present study, the injection of digibind decreased
{ 3 min. (Figure 2). In contrast, MAP was decreased blood pressure in ACTH-treated but not sham treated
by preimmune sheep IgG (Fab)2 fragment at 35 { 3 min rats. However, both preimmune sheep IgG and sheep
and reached a minimum at 45 { 5 min (Figure 1). MAP IgG (Fab)2 fragment also decreased blood pressure in
decreased after preimmune sheep IgG at 1 { 0.2 min the rat, although the time courses were different. For
and reached a minimum after 1 { 0.3 min. The effect example, preimmune sheep IgG started to drop MAP
lasted 40 { 3 min (Figure 3). earlier and lasted longer than did digibind, and the

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AJH–MARCH 1997–VOL. 10, NO. 3, PART 1 HEMODYNAMICS OF DIGIBIND IN ACTH-HYPERTENSION 335

effect on MAP of preimmune sheep IgG (Fab)2 frag-

ment treatment was slower than that of digibind injec-
tion. The effects of digibind might reflect the nonspe-
cific hypotensive side effects of immunoglobulin
preparations, which may be effective in the hyperten-
sive ACTH group but not in normotensive sham con-
trols.
Hypotension is a well documented side effect of
intravenous administration of plasma protein prepa-
rations.12 Different plasma protein preparations may
induce different blood pressure responses. In contrast
to the present study, Mann et al5 reported that neither
preimmune IgG nor preimmune Fab fragment (Boeh-
ringer, Mannheim, Germany) decreased blood pres-

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sure in the rat. However, in the same study, digibind
(60 mg/rat, intravenously) did not alter blood pres-
sure in DOCA-salt hypertensive rats. In contrast, us-
ing 0.9% NaCl as digibind vehicle, a recent study7
reported that in DOCA-salt hypertensive rats digibind
(10 mg/kg, intravenously) lowered blood pressure
significantly without change of sodium pump activity.
However, neither preimmune sheep IgG nor preim- FIGURE 3. Mean arterial pressure (MAP) in sham plus digi-
mune Fab fragment were used in that study. It is well bind (n Å 6) and ACTH plus digibind (n Å 8) treated rats.
Statistics compare precontrol (0) with treatment time (min) (*P
documented that digoxin-binding antibodies reverse
õ.05 and **P õ.01).
the effect of endogenous DLS on the sodium pump. 13
These data raise questions about possible vasodilatory
contaminants in digibind. Accordingly, caution is
needed in interpretation of the digibind infusion data. In summary, the acute administration of digibind
Previous studies3,4,6,7 without appropriate controls caused a pronounced, long-lasting blood pressure de-
should be reevaluated. crease in ACTH hypertensive rats, but had no effect
on blood pressure of sham treated rats. These data
are consistent with the notion that digitalis-like sub-
stances may play a role in ACTH induced hyperten-
sion, but the evidence that both preimmune sheep
IgG and IgG (Fab)2 fragment also decreased blood
pressure in rats suggests that studies of digibind infu-
sion should be interpreted with great caution.
ACKNOWLEDGMENTS
This work was supported by the Clive and Vera Ramaciotti
Foundation, a General Development Grant from the Univer-
sity of New South Wales, and the National Health and Medi-
cal Research Council of Australia. ACTH was generously
provided by Ciba-Geigy Australia Limited.

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