TOXICOLOGY
AND
APPLIED
Subacute
PHARMACOLOGY
Digoxin
35, 283-301 (1976)
Toxicosis
in the Beagle
Dog
R. H. TESKE,S. P. BISHOP,~H. F. RIGHTER,AND D. K. DETWEILER~ Division of Veterinary Medical Research, Food and Drug Administration, Beltsville, Maryland 20705, College of Medicine, Ohio State University, Columbus, Ohio 43210, and School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104 Reeeived June 16,1975; accepted September 3,1975
SubacuteDigoxin Toxicosisin theBeagleDog. TESKE, R. H., BISHOP, S.P., RIGHTER,H. F., ANDDETWEILER, D. K. (1976). Toxicol. Appl. Pharmacol. 35, 283-301. Digoxin was administerediv to healthy beagledogsat dose levelspreviously shownto inducesustainedtoxicosisand to permit survival over a 2-weekperiod. Treatment regimensof both 5 and 7 days/weekwere employed.Electrocardiographic(ECG) responses, clinical pathology, and serum digoxin concentrationswere monitored, and, at necropsy, tissues were examined for pathological changes.The development of digoxin toxicosisamongtreated dogswasextremely variable and did not correlate well with the amount of digoxin administered. However, correlation betweenthe degreeof toxicity and serumdigoxin concentrationsappeared to be good. The results indicate that of the variables monitored at the samplingtimesemployed,ECG responses werethe mostsensitive,prompt, and consistentindicators of digoxin toxicity whenmoderately toxic serum digoxin concentrations (2.5-6.0 rig/ml) were attained in normal beagle dogs.Elevationsin activities of serumcreatininephosphokinase(CPK) and lactic dehydrogenase(LDH) were lessreliable. At severelytoxic serum digoxin concentrations(above6.0 rig/ml), elevatedCPK and LDH activities appearedearly and persistedfor severaldaysuntil the meanserumdigoxin concentrationfell below 6.0 rig/ml. An unexpectedfinding wasevidenceof compromisedrenal function asindicated by elevatedserumcreatinine and blood nitrogen concentrations and by alterations in serumelectrolytes. Thesechangeswere associatedwith histopathological evidence of renal damage in all dogs.
Digitalis toxicosis in humans has been estimated to occur in 6-35 % of treated patients (Chung, 1969; Giuffra and Tseng, 1952; Resnick, 1964; Rodensky and Wasserman, 1961,1964; Sodeman, 1965; Smith and Haber, 1973b), and the mortality rate of 3-21x among those intoxicated remains a major medical problem. Despite this prevalence and importance, there have been relatively few investigations of subacute and chronic digitalis intoxication in experimental animals (Buchner, 1934; Dearing et al., 1943a,b; Hu et al., 1936; Kyser et al., 1946; Selye, 1961). Digitalis therapy was introduced by Sir William Withering in 1785, Iong before preclinical evidence of efficacy and safety of drugs was required. McMichael(l972) has pointed out that this epoch-making contribution to medical practice would not have passedpresent efficacy criteria. Currently, there is ample experimental evidence for the 1 College of Medicine, Ohio State University, Columbus. z School of Veterinary Medicine, University of Pennsylvania, Philadelphia. Copyright 0 1976 by Academic Press, Inc. 283 All rights of reproduction in any form reserved. Printed
in Great
Britain
284
TESKE ET AL.
beneficial effects of digitalis glycosides. However, knowledge of chronic toxic effects is confined largely to observations on human patients in whom toxicity is accidental and ameliorated at the earliest possible stage. Studies in animals have been chiefly concerned with acute effects (Detweiler, 1967). In a previous investigation (Fillmore and Detweiler, 1973), an iv digoxin dosage regimen was established for beagle dogs which permitted sustained toxicosis with survival over a 2-week period. The present study was initiated to investigate further the manifestations of subacute digoxin toxicosis in healthy beagle dogs. The aim was to compare methods of detecting digoxin toxicosis and relate them to serum digoxin concentrations and necropsy findings. METHODS Thirty-four purebred beagles3 (16 males, 18 females), 9-36 months old, were used in the study. Six dogs served as controls and the remaining 28 dogs were assigned to test groups; baseline observations were made on each dog. The dogs were housed individually in cages, and food and water were available ad libitum. Digoxin was administered iv via the cephalic vein, employing a loading dose on the initial day of treatment followed by daily maintenance doses. The loading dose consisted of an initial dose of 50 pg/kg followed 1 hr later by a second dose of fiOpg/kg. Four hours after the initial dose a third dose of 25 ,ug/kg was administered, and maintenance doses ranging from 15 to 35 ,ug/kg were given iv once daily thereafter. The severity of toxicosis, asjudged by physical and electrocardiographic signs, was evaluated daily and appropriate adjustments in maintenance doses were made. Occasionally, daily maintenance doses were omitted when it was considered that administration might result in death. Treatment regimens of 5 days/week (14 test and 4 control dogs) and 7 days/week (14 test and 2 control dogs) were employed. When digoxin was given 5 days/week, a modified loading dose (50 pg/kg) was given on the first day of each succeeding 5-day treatment period. The following variables were monitored. General Observations
All dogs were observed daily for gross signs of toxicosis, appetite, general appearance, and behaviour. Body weights were recorded twice weekly. Eiectrocardiography (ECG)
ECG was recorded by using a rectilinear ink recorder and the McFee orthogonal lead system (McFee and Parungao, 1961). The dogs were positioned in dorsal or right lateral recumbency; alligator-clip electrodes were saturated with 50% ethanol and attached to the skin. The ECGs were interpreted according to the criteria for ECG evidence of digoxin action previously reported (Fillmore and Detweiler, 1973). The configuration of the ECG in the Y lead of the McFee orthogonal system is comparable to Einthoven bipolar lead II; the McFee Z lead resembles somewhat VlO of the canine unipolar chest leads ; and the McFee X lead is recorded between a pair of electrodes on either side of the thorax, i.e., this lead is related to the canine Wilson system thoracic unipolar leads recorded from the right (CVSRL) and left (CV6LL, CV6LU) thorax (Detweiler 3 From the beagle colony of the Division of Veterinary Medical Research, Food and Drug Administration, Beltsville, Maryland.
SUBACUTE DIGOXIN TOXICOSIS IN DOGS
285
et al., 1968; Fillmore and Detweiler, 1973; McFee and Parungao, 1961). Thus the thoracic leads of the two systems, although related, are not identical. The signs of digoxin toxicosis previously classified by Fillmore and Detweiler (1973) as presumptive or reliable for the Wilson lead system were modified for the McFee system as follows. Presumptive signs.T-wave flattening or reversal : lead X, Z; S-T segment deviation, less than 0.15 mV; wandering pacemaker; variable P-R interval without dropped beats; P-R interval increased 20 % over control but less than 150 msec. Reliable signs. Sinus tachycardia greater than 200; sinus bradycardia less than 50; 1” atrioventricular block (P-R interval greater than 150 msec); incomplete atrioventricular block with dropped beats (second degree); atrioventricular dissociation; paroxysmal atria1 tachycardia (PAT) with or without block; ectopic atria1 beats; fusion beats ; interventricular block; S-T segment deviation greater than 0.15 mV; ventricular ectopic beats. The numerical scoring system for grading intensity of the ECG effects of digoxin, proposed earlier for the Wilson lead system (Fillmore and Detweiler, 1973), was used to quantify the ECG effects observed with the McFee lead system. This scoring system provides for quantification of ECG effects (a) on a daily basis (digoxin toxicosis score); (b) for the duration of the study period (total digoxin score) ; and (c) average effect over the study period (digoxin toxicosis index; DTI). The ECG scoring system, although arbitrary, was designed to reflect the degree of ECG abnormality observed in records taken on any given day. A score of 0 indicated that none of the ECG signs listed above was observed; 1 indicated one presumptive sign, and 2 indicated two or more presumptive signs. A score of 3, 4, or 5 indicated that 1, 2, or 3 reliable signs were observed, respectively. As in the previous work (Fillmore and Detweiler, 1973) with this scoring system, more than three reliable signs of digoxin toxicosis were not observed in any set of ECGs from a single dog. Hematology and Serum Chemistry Determinations
Blood samples for hematology and serum chemistry determinations were collected from 29 dogs (24 digoxin-treated and 5 control dogs) two to four times during the predose control period and at 1-3-day intervals during the treatment period. Additional baseline control values were tabulated from 168 normal beagle dogs in the same colony (Table 1). Complete blood counts and serum chemistry determinations were performed by an independent commercial laboratory. Serum chemistry determinations were performed by automated techniques and included total protein, albumin, uric acid, blood urea nitrogen (BUN), creatinine, glucose, sodium, potassium, chloride, calcium, inorganic phosphorus, alkaline phosphatase, serum glutamic-oxaloacetic transaminase (SGOT), lactic dehydrogenase (LDH), and creatinine phosphokinase (CPK). Serum digoxin concentrations of 20 dogs in the study were determined by radioimmunoassay; the blood samples for these determinations were collected at I-Zday intervals, in the morning, 24 hr after the previous dose. Necropsies
During the study, five dogs died from digoxin toxicity and a necropsy was performed within 12 hr of death. Four treated dogs and one control dog were killed by electro4 Schwarz/Mann,
Orangeburg, New York.
VALUES
0 Division
of Veterinary
Medical
Hematology Erythrocytes (x 106/mm3) Leukocytes (x 103/mm3) Hemoglobin (g/100 ml) Hematocrit (%) Mean corpuscular hemoglobin concentration, (%)
Research
76 76 77 23 23
Ekltsville,
34.6 k 2.2
43 Colony,
6.82 + 12.6 + 15.97 + 47.6 +
Maryland.
32-45
42
56 79 81 81
91 92 90 29 28
90 88 90 90 89 63 90 61 88
Number of
DETERMINATIONS
18-70 2.5-6.2 6.7-l 1.3 142-168 4.5-5.3
4.6-7.6 1.2-3.3 55-120 lo-24 0.34.9 0.6-l .o 20-177 37-260 25-300
Range
1
1.09 5.21-10.63 4.0 7.3-24.3 1.67 12.3-19.5 5.4 36-55
12.8 1.01 0.83 6.4 0.25
57 74 73 75
f. + + f. +
47.4 4.28 10.16 151.2 4.96
77 78 77 59 77 56 76
0.61 0.80 21.2 5.4 0.16 0.18 31.4 50.4 65.6
Mean *SD
TABLE HEMATOLOGY
Males
AND
6.47 + 1.90 rt 87.9 + 16.2 f 0.55 k 0.71 + 67.8 + 93.7 + 117.3 +
of dogs
CHEMISTRY
Beagle
FOR SERUM
Serum chemistry Total protein (g/100 ml) Albumin (g/100 ml) Glucose (mg/ 100 ml) Blood urea nitrogen (mg/lOO ml) Uric acid (mg/lOO ml) Creatinine (mg/lOO ml) Alkaline phosphatase (mu/ml) Creatinine phosphokinase (mu/ml) Lactic dehydrogenase (mu/ml) Serum glutamic-oxaloacetic transaminase (mu/ml) Inorganic phosphorus (mg/lOO ml) Calcium (mg/ 100 ml) Sodium (mequiv/liter) Potassium (mequiv/liter)
Determination
BASELINE
15.0 0.99 0.69 5.0 0.36
0.57 0.80 24.8 5.3 0.18 0.10 39.6 39.6 84.4
AND
25-108 2.4-6.2 9.4-11.5 136-161 4.1-5.3
4.8-7.7 1.4-3.7 40-140 l&32 0.4-1.1 0.5-1.0 30-270 30-400 25-370
Range
FEMALE
34.6 + 1.4
32-37
6.69 &- 0.97 4.93-10.40 11.2k4.3 5.5-28.0 16.07 + 1.67 12.0-19.0 37-62 48.0 f 4.9
45.3 + 4.23 f 10.35 + 151.3 + 4.80 f
6.40 f 2.07 + 91.8 + 17.1 f 0.52 + 0.69 2 74.6 f 85.6 f 124.5 k
Mean fSD
Females
IN MALE
85
113 153 154 156
167 168 167 52 51
168 166 167 168 166 122 167 117 164
Number of dogs
BEAGLES”
Mean *SD
1.03 4.1 1.61 5.2
13.6 1.00 0.77 5.6 0.32
34.6 f 1.8
6.76 + 11.9 f 16.16 f 47.8 f
46.3 + 4.26 + 10.27 + 151.3 + 4.87 f
6.43 + 0.59 1.99 + 0.80 90.0 If: 23.2 16.6 * 5.4 0.53 + 0.17 0.70 + 0.14 71.5 + 36.1 89.5 + 45.1 121.2 k 76.1
Total
z 2 ’
2 $j
E
SUBACUTE
DIGOXIN
TOXICOSIS
IN DOGS
287
cution, and the tissues were preserved in 10 % phosphate-buffered formalin. The remaining dogs were anesthetized with sodium pentobarbital, the thorax was opened, and the heart was retrogradely perfused via the aorta with isotonic phosphate-buffered saline followed by 2% phosphate-buffered glutaraldehyde at a pressure of approximately 100 Torr. Complete necropsies were performed on all animals and representative sections from all body organ systems were fixed in 10% phosphate-buffered formalin. Fixed tissues were taken from multiple preselected areas of the myocardium to include all major anatomic components of the heart and from other major organs of the body, embedded in paraffin, sectioned at 6-pm thickness, and stained with hematoxyhn and eosin. Heart sections were also stained with Gomori’s aldehyde fuchsin trichrome stain. Studies on 24 dogs were conducted at the Division of Veterinary Medical Research (DVR) laboratories, Beltsville, Maryland. Studies on the remaining 10 dogs were conducted at the Ohio State University, Columbus, Ohio. RESULTS
Physical Examination The digoxin-treated dogs generally showed signs of CNS depression with decreased activity; they also had a decreased food and water intake, and a mild loss of weight during the treatment period. Vomiting often occurred during the 1 or 2 days after the loading or modified-loading dose but did not persist. Diarrhea was not present in any dog. Dehydration was usually very mild; however, in one trial with four dogs, dehydration became severe during the period when the serum digoxin concentration was above 6 rig/ml but became less severe with decreased serum digoxin. The severity of these signs appeared to be well correlated with the digoxin concentration in serum. Representative samples of these changes in two dogs are illustrated in Fig. I. In all dogs, heart rate was determined from the ECG each day, and during the control period it ranged from 90 to 130 beats/min after the dogs became accustomed to having ECGs recorded. There was a sharp drop in heart rate to 60 % of control values (50-90 beats/min) by 6 hr following the loading dose. By 24-48 hr the heart rate had returned to control predose values. During the periods of most severe toxicity, the heart rate was elevated to 130-190 beats/min (Fig. 1). Body temperature was inversely related to the concentration of serum digoxin and other related signs of toxicity. The temperature was as much as 3°F lower than control predose temperature in the dogs most severely affected. Temperature decreased by approximately 1°F when serum digoxin concentrations exceeded 4 rig/ml and decreased further at more elevated concentrations. Serum Digoxin Serum digoxin concentrations varied considerably among individual animals. In eight dogs of the group given digoxin 5 days/week in which serum digoxin was determined, the concentration generally remained between 1.5 and 5.0 rig/ml by 4 days after the loading dose. After 2 days without treatment, however, the serum digoxin concentration was less than 1.2 rig/ml, and with the modified loading dose, gradually rose to 2.0-3.5 ng/rnl with 4 additional days of treatment. The same pattern held for two dogs that were treated for a third week following a second 2-day period with no dose; the concentrations finally reached 2.5 and 4.0 rig/ml on the seventeenth day of the study.
288
TESKE
ETAL.
Dogs treated 7 days/week with digoxin generally had higher digoxin concentrations in serum than those treated 5 days/week; these concentrations were sustained unless the dose was reduced. The difficulty in establishing a stable toxic serum concentration of digoxin is illustrated in Figs. 2 and 3. The dogs whose concentrations are shown in Fig. 2 received digoxin at 25 pg/kg on Day 1 following the loading dose on Day 0 and maintained very high toxic concentrations of digoxin, between 8 and 10 rig/ml, until the DIGOXW TOXICITY IN THE DOG 89V
59x
IO -
- I25
Ge-
‘98
4-
0:- g E-
c t 4’
600400-
6
4
IO
I2
I4
I6
lb
i
6
600,
bFs3
4
;a *5 Q-
IO
1
- 1000
200-
.
C
F? az E’
2
4
6
6
IO
I2
I4
I6
C
I8
160
102
100
wo 101 ,
2
I
4
I
6
I
6
1
0
1
I
I
IO
I2
I4
I6
I6
‘60 I60 120 zi 30
C
2
4
6
6
IO
I2
I4
I6
16
600 400 200
t :w
C
2
4
6
6
W
3
.
C
ze 85
I25
‘8
2
4
6
6
IO
12
I4
I6
I6
i[?K C 600 400-l
2
4
1
E 6
6
IO
-3 fE zg
rm
k400
2oo~200
DAY9 FIG. 1. Correlation of toxicosis indices with serum digoxin concentration administered for two separate dogs. Digoxin was given 7 days/week.
and dose of digoxin
dose was reduced to 10 pg/kg or omitted entirely during the second week of treatment. Although the dogs in this trial (Fig. 2) responded very similarly, four dogs in a subsequent trial (Fig. 3) had marked variations in their response to iv digoxin administration. In this second trial, no dose was given on Day 1 following the loading dose, and in two of the dogs (59X and 64X, Fig. 3), there was a slow gradual increase of serum digoxin within the toxic range above 2.5 rig/ml while the dose was maintained relatively constant at 25-35 pg/kg. In dog 63X, serum digoxin rapidly reached severely toxic levels, above 6 rig/ml, at the 25-,ug/kg dose level. In contrast, dog 61X had very low concentrations of serum digoxin which only reached mildly toxic levels after 8 days of treatment in spite of a dose increase to 35 ,ug/kg.
SUBACUTE
DIGOXIN
SERUM
MGOXIN
TOXICOSIS AND DIGOXIN
289
IN DOGS DOSE
FIG. 2. Serum digoxin concentrations are indicated for four separate dogs by the connected symbols and the total dose administered iv to each dog by the nonconnected symbols. Injections were started on Day 0 and were given 7 days/week. Serum digoxin concentrations for each day were determined on samples collected prior to administration of digoxin.
ll?zcil Ik/ M SERUM
DIGOXIN
AND DBOXH
DOSE
64X
59x
lo 6 6 4 2 0
126
IO
126
loo
6
loo
76
6
75
60
4
60
26
2
25
0
0
2
4
6
6
IO
024660
63X
IO
126
IO
I25
loo
6
100
I6
6
76
4
SO
4
60
2
2s
2
26
6 6
0
0 0
2
4
6
6
IO
0
2
4
6
6
IO
DAYS FIG.
3. Serum digoxin concentration
and digoxin dose for four separate dogs treated 7 days/week.
Electrocardiograms
Many alterations in ECGs were encountered during the course of the experiment. The most frequently found changes included first and second degree heart block (prolonged P-R interval with or without dropped beats), ventricular extrasystoles or
0 1 0 0 0 0 0 0 0 0 0 -0 -0 -
0 0 1 0
Digoxin 5 days/ week 21U (Control) 80T (Control) 12U (Control) 81T (Control)
31T 38U 86s 79T 31u 42U 74T 82T 58V 60V 94u 61V 62V 63V
0
Dog number
DAILY
0
1 3 0 4 3 1 1 3 0 2 1
0 0 0 0
1
DIG~XIN
2 3 1 0 0 2 1 0 3 3 2 1 2 3
0 0 0 0
2
1 3 3 0 0 1 0 1 2 3 3 3 3 3
0 0 0 0
3
TOXICITY
2 1 1 1 1 0 3 3 3 1 2 3 4 1
0 0 0 0
4
SCORES
-
-
-
-
-
-
-
-
-
6
-
5
IN BEAGLE
TABLE
2
13 3 3 3 0 2 3 FD 3 1 1 0 3 3
0 0 10 0
7
3 2 1 3 0 4 3
3 1 2 3 3 3
2 4 4 4
0 0 11 0
9
3 3 4 11 12 3
0
0 0
8
day
2 3 3 3 4 3
3 4 4 5 11--l 3 3
0
0 0
10
Experiment
--2 FD --3
--3 --0
3 3 s s s 0 4 3
-----0 ---
---0
13
DIGOXIN
--0 -----0
12
WITH
3 4 3 FD
0 0 l--l 0
11
DOGS FOLLOWING TREATMENT TOXIC DOSAGE LEVELS’
14
4
4
3
3 3 3
3 3 3
4
5 4 4
0 0 1 0
16
3 4 4
0 0 0 0
15
ADMINISTERED
17
3
3
3 3 3
4 4 FD
AT
3
2
3 3 3
5 4
18
2.69 3.14 2.72 2.62 1.50 2.14 2.28 1.75 2.25 1.87 2.12 2.08 3.38 3.00
DTI
1.35
-3.66
4 3 4
:
3 4 4 4
-
2.00
4 4 4 4 3 3
-
2.53
3 4 4 4 3 3
3 3 3 1 1 3
0
2.53
4 4 4 4 3 4
3 3 3 3 1 3
--
3.15
4 4
3 4
3 5 2 3 1 3
2.66
-
5 4
FD
__-
-
55 44
-3 -4 -3 -3
3.57
3
1.50
3.29
5 4
4 3 3 3 11
-
3.50
3.14
5 4
3 4 2 3
-
3.62
2.83
5 -
3 3 2 4 0
-
3.28
2.60
3 3 1 3 3
0
3.28
2.64 3.35 2.00 1.78 1.14 1.44 3.25 3.33 3.64 3.38 2.50 3.12 2.11 2.55
0 0
2.39
gx
$j z
%
,,,,,
,,,
,,
1.35
2.00
2.00
3.00 2.66
2.75
2.91
,”
3.25
I,,
3.20
/I,
? g 2
4
3 2 4 ---
----
0 0 3 3 2 3
---
3 3
0
1.86
’ The digoxin toxicity scores are a subjective numerical scoring system of presumptive (l-2) and reliable (3--S) signs of toxicity (see Methods for the criteria for electrocardiographic evidence of digoxin toxicity). DTI, digoxin toxicosis index; FD, found dead; S, sacrificed.
1.28
0 3 4 3 3
3 3
0
1.86
8w
0
Mean
0 3 lll-10 110 0 3 3 3 0 112 14 lll-113
0
1.64
2.58
00 14 02 01 02 00 04 03 02 00 00 00 00 00
-0 o------
0
36 36T 68T 98U 99u 97u 45w 54w 89V 9ov 59x 63X 61X 64X
Mean Digoxin 7 days/ week 94V (Control) 96V (Control)
292
TESKE ET AL. DIGOXIN
TOXICITY (45W) McFEE
IN THE LEAD
DOG
2
lrec CONTROL
I DAY
FIG. 4. McFee lead Z electrocardiogram during control period and 1 day after the loading dose. Ventricular extrasystoles are present on the Day 1 recording. oIoox#
TOXICITY IN THE DDG (59X) McFEE LEAD Y
CONTROL
SERUM DIGOXM 80 rig/ml
2 DAYS
SERUM DlOOXlN * 3.0 rig/d
7 DAYS
SERUM DIGOXIN = 6.4 rig/ml
SERUM IO DAYS 5. McFee lead Y electrocardiogram during week. At 7 and 10 days there is second degree heart ~asuperimposed on the preceding T-wave, but are FIG.
DlGOXlN=lO rig/ml subacute digoxin toxicity in a dog treated 7 days/ block with dropped beats. On Day 10 the P-waves clearly identified on the beat following a dropped
SUBACUTE
DIGOXIN DEOXW
7 CMYS SERUM
293
IN DOGS
TOXlClTY IN THE DOG (SW McFEE LEAD 2
IO DAYS SERUM
DlG4D.Otqhl
17 DAYS
TOXICOSIS
SERUM
DIG= 10.0 rig/ml
DlG=5.1 rig/ml
FIG. 6. McFee lead 2 electrocardiogram during subacute digoxin toxicity in a dog treated 7 days/ week. There is bradycardia at 6 hr, S-T segment depression at 7 days, second degree heart block with dropped beats at 10 days, and paroxysmal A-V dissociation with sinus block and ventricular extrasystoles at Day 17.
d LL
III*
LL
IImv
1
Irr
L.
. 4% I45
I70
220
250
FIG. 7. McFee lead X, Y, and Z electrocardiograms from a dog (59X) that developed signs of severe toxicity during the course of digoxin treatment 7 days/week. One-millivolt standards are marked for each lead; X2 = 1 mV is twice the amplitude marked by the bar. Paper speed was identical (30 mm/set) for all tracings. Changes in the wave form of the QRS complex and T-wave (ventricular aberration) are apparent with increasing serum level of digoxin and prolongation of the P-R interval.
escape beats, deviation of the S-T segment, and alterations in the QRS and T-wave form. The daily digoxin toxicity score based on the finding of presumptive or reliable ECG signs of toxicity and the DTI for each dog are listed in Table 2. Ventricular ectopic
294
TESKE ET AL. DIGOXIN DAY SERUM
DIG
C (0)
6h (7.0)
I (4.6)
TOXICITY IN THE DOG (61X1
2 (I.8 i)
3 (1.3)
4 (I.
7 2.0)
8 (2.9)
9 (3.2)
IO 14.~
4J
II
1
m
-fI T
I II5
I II5
120
120
120
125
160
FIG. 8. McFee lead X, Y, and Z electrocardiograms from a dog (61X) treated 7 days/week and having only mild to moderate digoxin toxicosis. Wave form changes are less pronounced than those in Fig. 7.
beats, when present, usually occurred during the first week of treatment and resulted in a digoxin toxicosis score of 3 or 4 (Fig. 4). The P-R intervals became progressively prolonged in nearly all dogs, with dropped beats occurring during the second week of treatment in many dogs (Fig. 5). Other less common arrhythmias sometimes found in instances of severe toxicity (Fig. 6) are listed in Methods (Electrocardiography). Deviations of the S-T segment were occasionally found but seldom were greater than 0.25 mV. Changes in the wave form pattern of the QRS complex and the T-wave (ventricular aberration) were frequent findings (Figs. 7 and 8). Hematology and Clinical Chemistry
Total erythrocyte count, hemoglobin, packed cell volume, red cell indices, and total and differential leukocyte counts remained within normal limits throughout the study, except for four dogs (45W, 54W, 89V, and 90V) that became severely dehydrated with resulting hemoconcentration associated with severely toxic concentrations of digoxin in serum. Mild fluctuations in packed cell volume seen occasionally in other dogs were associated with the state of hydration but were not outside normal limits. Alterations in serum CPK and LDH activities and in BUN, creatinine, sodium, chloride, and potassium occurred in dogs with digoxin toxicosis. All other clinical chemistry determinations remained within normal limits. Tables 3 and 4 list the range of values for those determinations with altered values, together with the median for each group and the number of determinations that exceeded normal limits for each experimental day. The median is given since the curve is not normally distributed but skewed to the right. Normal baseline values for dogs in the beagle colony from which the dogs in this study were derived are given in Table 1. Representative examples of the day-to-day fluctuations of the various indicators of digoxin toxicity are shown jn Fig. 1 BUN values were elevated to over 25 mg/lOO ml in 21 of 28 dogs examined, including all of the dogs treated 7 days/week. Serum creatinine was elevated up to 8.1 mg/lOO ml
-
(60)
40-280 (75) 30-130 (75) 30-145 (90) 3@230 (110) 30-120 (50) 30-380 (50) 40-350
(80)
40-100 (70) 50-150
Range
CPK (mu/ml)
215
215
O/5
219
o/9
o/9
l/10
o/10
o/10
Xln
OF SUBACUTE
LDH (mu/ml>
40-1005 (105) 45-780 (90)
(210)
100-700 (390) 105-660 (350) 80-810 (405) SO-450
(210)
95-500 (143) 651220 (155) 55-670
215
2/5
215
6/9
5/9
6/9
2/10
O/l 0
o/10
X/n
TOXICITY
Range
DIGOXIN
TABLE DOSE)
3
23-46 (44
(36)
17-54
(23
10-17 (15) 1O-28 (17) 14-55 (19) 12-25
15-100 (20)
(21)
10-18 (15) 18-28
Range
415
415
O/5
t/9
t/9
O/9
2/10
l/10
o/10
X/n
BUN (mg/lOO ml)
(~-DAYS/WEEK
CHEMISTRY
(0.8)
0.7-1.0 (0.7) 0.6-1.0 (0.8) 0.7-l .2
(0.6)
Oh-I.0
(0.6)
0.5-l .o
(0.6)
0.660.9 (0.7) 0.7-0.9 (0.8) 0.6-5.5 (0.7) 0.6-1.0
Range
l/5
o/5
o/5
o/9
o/9
O/9
l/10
o/10
O/10
Xln
Creatinine (mg/lflO ml)
ON SERUM
DOGS’
126-158 (141)
(136)
140-151 (146) 14&150 (146) 144153 (149) 130-153 (145) 130-150 (1W 129-149
(146)
146-158 (151) 141-152
Range
215
315
2/5
2/9
o/9
o/9
o/9
O/l0
O/10
X/n
Sodium (mequiv/liter)
OF BEAGLE
4.4-5.1 (4.8) 3.3-5.3 (4.7) 4.1-4.8 (4.5) 3.8-5.3 (4.4) 4.2-5.3 (4.9)
(4.6)
4.4-5.2 (4.8) 3.6-5.3 (4.7) 4.4-5.2 (4.9) 4.3-5.5
Range
O/5
215
l/5
219
o/9
l/9
O/9
l/10
O/10
X/n
Potassium (mequiv/liter)
’ CPK, creatinine phosphokinase; LDH, lactic dehydrogenase; BUN, blood urea nitrogen; X/n, number of values which exceed 95 % confidence limits of normal values/number of observations for that date. Normal values are derived from Table 1 and are: CPK < 185 mu/ml, LDH < 275 mU/ml, BUN < 27 mg/lOO ml; sodium 140-160 mequiv/liter; potassium 4.2-5.4 mequiv/liter. Median values are given in parentheses.
18
16
14
11
9
7
4
2
0
Day
EFFECT
318
w3)
97-305
(160)
91-280
076)
70-508 (180) 120-585 (170) 10&490
(165)
6/ 10
l/9
3/12
6/l 3
5/ 13
70-l 187 7/14 (170) 40-1070 6/14
(185)
o/14
40-171 (140) 90-230 (115) 90-417
3/10
X/n
Range
(210)
35-475 (185) 80-445 (190) 41-700 (147) 55-600 (145) 56-820 (131) 58-550 (150) 52-700
(136)
lW450 (130) 104-180
(160)
75-215
Range
LDH (mUhI)
S/10
219
2112
6/l 3
3/l 3
5114
2114
O/8
2/10
O/14
X/n
15-120 (37)
(32)
12-100 (40) 15-191 (4-v 15-87
(20)
13-165 (225) 13-165
(2%
9-24 (14) lo-21 (13) 9-82 (30) 14-202
Range
6/10
519
6/12
9/13
6/13
6/14
7/14
418
o/10
o/14
Xln
BUN (mg/lOO ml)
(0.8)
O-6-2.6 (1.0) 0.5-1.6 (1.0) 0.6-2.4 (1.1) 0.7-3.2
W3)
0.5-2.1
(0.8)
0.6-5.6 (1.1) 0.7-8.1
(0.8)
0.6-0.9 (0.7) 0.6-1.1 (0.7) 0.4-2.8
Range
3/10
519
6/12
6/l 3
5/12
6/14
8/14
3/8
l/10
O/14
X/n
Creatinine (mg/lOO ml)
123-159 (135) 119-175 (132)
(128)
121-152 (142) 48-150 (139) 39-l 50 (145) 114-147 (140) 116142
(148)
149-153 (150) 146151 (149) 142-152
Range
7/10
6/9
6/11
8/13
4/l 1
4/8
4/11
O/S
O/8
o/14
X/n
Sodium (mequiv/liter)
3.8-5.5 (5.0) 3.47.7 (5.1) 3.9-6.8 (4.8) 4.0-5.6 (4.9) 3.7-5.2 (4.7) 3.3-5.8 (4.3) 3.2-5.7 (4.3)
(4.6)
4.6-5.2 (4.9) 4.3-5.2 (5.0) 4.4-5.0
Range -.___
8/10
519
2/l 1
3/l 3
3/13
4/l 1
2/l I
O/8
O/8
O/14
X/i1
Potassium (mequiv/liter)
4 DOSE) ON SERUM CHEMISTRY OF BEAGLE DOGS"
-
61, 82
(56)
71-l 10 (75) 32-97
19,106
107-l 12 (110) 99-l 06 (100) 64/106 (87) 20-l 10 (59) -
Range
212
717
314
l/2
517
718
O/8
X/11
Chloride (mequiv/liter)
n CPK, creatinine phosphokinase; LDH, lactic dehydrogenase: BUN, blood urea nitrogen; X/n, number of values which exceed 95 s,; confidence limits ot normal values/number of observations for that date. Norma1 values are derived from Table 1 and are: CPK < 185 mu/ml, LDH 4 275 mu/ml, BUN < 27 mg/lOO ml; sodium 140-160 mequiv/liter; potassium 4.2-5.4 mequiv/liter; chloride 100-I 14 mequiv/liter. Median values are given in parentheses.
18
15
12
10
8
Day
CPK (mu/ml)
TABLE EFFECT OF SUBACUTE DIGOXIN TOXICIIY(~-DAYS/WEEK
Q k .
;;t g
E
SUBACUTE
DIGOXIN
TOXICOSIS
297
IN DOGS
in parallel with the increased BUN. In dogs with the most severely elevated BUN (over 100 mg/lOO ml), there was frequently a mild increase in packed cell volume associated with decreased water intake and dehydration. Serum sodium and chloride were moderately to severely reduced in several dogs concomitant with the elevated BUN and were especially marked in those dogs that had the most severe dehydration. Serum potassium was mildly elevated in several dogs, although, in a few, serum potassium was lower than normal (Tables 3 and 4). Serum CPK and/or LDH activities were mildly to moderately elevated at some time during treatment in 7 of the 10 dogs examined that were on the 5-day/week dose and in 11 of 14 dogs on the 7-day/week dose. In many dogs the increased activity of serum enzymes conincided with the highest serum digoxin concentrations and other signs of toxicity, but this was not always the case. In some dogs there appeared to be no obvious correlation of elevated serum enzyme activity with other signs of toxicity; CPK and LDH activities were usually both elevated at the same time, but sometimes one was elevated while the other remained within normal limits. Because isoenzymes of CPK or LDH were not determined, it is not possible from the clinical chemistry data to identify which body organ was injured to release them. However, the only organs with lesions found at necropsy were the heart and kidneys (vide i&z). Table 5 summarizes the changes that occurred with digoxin administration. TABLE 5 SUMMARY OF CHANGES OCCURRING WITH DIGOXIN ADMINISTRATION CORRELATED WITH THE DEGREE OF TOXICOSIS AND SERUM DIGOXIN CONCENTRATIONS
Nontoxic or mild toxicity
Moderate toxicity
Severe toxicity
Sodium (mequiv/liter) Chloride (mequiv/liter) Potassium(mequiv/liter) CPK, LDH
K2.5 Normal Normal Normal Normal
AND
APPLIED
Subacute
PHARMACOLOGY
Digoxin
35, 283-301 (1976)
Toxicosis
in the Beagle
Dog
R. H. TESKE,S. P. BISHOP,~H. F. RIGHTER,AND D. K. DETWEILER~ Division of Veterinary Medical Research, Food and Drug Administration, Beltsville, Maryland 20705, College of Medicine, Ohio State University, Columbus, Ohio 43210, and School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104 Reeeived June 16,1975; accepted September 3,1975
SubacuteDigoxin Toxicosisin theBeagleDog. TESKE, R. H., BISHOP, S.P., RIGHTER,H. F., ANDDETWEILER, D. K. (1976). Toxicol. Appl. Pharmacol. 35, 283-301. Digoxin was administerediv to healthy beagledogsat dose levelspreviously shownto inducesustainedtoxicosisand to permit survival over a 2-weekperiod. Treatment regimensof both 5 and 7 days/weekwere employed.Electrocardiographic(ECG) responses, clinical pathology, and serum digoxin concentrationswere monitored, and, at necropsy, tissues were examined for pathological changes.The development of digoxin toxicosisamongtreated dogswasextremely variable and did not correlate well with the amount of digoxin administered. However, correlation betweenthe degreeof toxicity and serumdigoxin concentrationsappeared to be good. The results indicate that of the variables monitored at the samplingtimesemployed,ECG responses werethe mostsensitive,prompt, and consistentindicators of digoxin toxicity whenmoderately toxic serum digoxin concentrations (2.5-6.0 rig/ml) were attained in normal beagle dogs.Elevationsin activities of serumcreatininephosphokinase(CPK) and lactic dehydrogenase(LDH) were lessreliable. At severelytoxic serum digoxin concentrations(above6.0 rig/ml), elevatedCPK and LDH activities appearedearly and persistedfor severaldaysuntil the meanserumdigoxin concentrationfell below 6.0 rig/ml. An unexpectedfinding wasevidenceof compromisedrenal function asindicated by elevatedserumcreatinine and blood nitrogen concentrations and by alterations in serumelectrolytes. Thesechangeswere associatedwith histopathological evidence of renal damage in all dogs.
Digitalis toxicosis in humans has been estimated to occur in 6-35 % of treated patients (Chung, 1969; Giuffra and Tseng, 1952; Resnick, 1964; Rodensky and Wasserman, 1961,1964; Sodeman, 1965; Smith and Haber, 1973b), and the mortality rate of 3-21x among those intoxicated remains a major medical problem. Despite this prevalence and importance, there have been relatively few investigations of subacute and chronic digitalis intoxication in experimental animals (Buchner, 1934; Dearing et al., 1943a,b; Hu et al., 1936; Kyser et al., 1946; Selye, 1961). Digitalis therapy was introduced by Sir William Withering in 1785, Iong before preclinical evidence of efficacy and safety of drugs was required. McMichael(l972) has pointed out that this epoch-making contribution to medical practice would not have passedpresent efficacy criteria. Currently, there is ample experimental evidence for the 1 College of Medicine, Ohio State University, Columbus. z School of Veterinary Medicine, University of Pennsylvania, Philadelphia. Copyright 0 1976 by Academic Press, Inc. 283 All rights of reproduction in any form reserved. Printed
in Great
Britain
284
TESKE ET AL.
beneficial effects of digitalis glycosides. However, knowledge of chronic toxic effects is confined largely to observations on human patients in whom toxicity is accidental and ameliorated at the earliest possible stage. Studies in animals have been chiefly concerned with acute effects (Detweiler, 1967). In a previous investigation (Fillmore and Detweiler, 1973), an iv digoxin dosage regimen was established for beagle dogs which permitted sustained toxicosis with survival over a 2-week period. The present study was initiated to investigate further the manifestations of subacute digoxin toxicosis in healthy beagle dogs. The aim was to compare methods of detecting digoxin toxicosis and relate them to serum digoxin concentrations and necropsy findings. METHODS Thirty-four purebred beagles3 (16 males, 18 females), 9-36 months old, were used in the study. Six dogs served as controls and the remaining 28 dogs were assigned to test groups; baseline observations were made on each dog. The dogs were housed individually in cages, and food and water were available ad libitum. Digoxin was administered iv via the cephalic vein, employing a loading dose on the initial day of treatment followed by daily maintenance doses. The loading dose consisted of an initial dose of 50 pg/kg followed 1 hr later by a second dose of fiOpg/kg. Four hours after the initial dose a third dose of 25 ,ug/kg was administered, and maintenance doses ranging from 15 to 35 ,ug/kg were given iv once daily thereafter. The severity of toxicosis, asjudged by physical and electrocardiographic signs, was evaluated daily and appropriate adjustments in maintenance doses were made. Occasionally, daily maintenance doses were omitted when it was considered that administration might result in death. Treatment regimens of 5 days/week (14 test and 4 control dogs) and 7 days/week (14 test and 2 control dogs) were employed. When digoxin was given 5 days/week, a modified loading dose (50 pg/kg) was given on the first day of each succeeding 5-day treatment period. The following variables were monitored. General Observations
All dogs were observed daily for gross signs of toxicosis, appetite, general appearance, and behaviour. Body weights were recorded twice weekly. Eiectrocardiography (ECG)
ECG was recorded by using a rectilinear ink recorder and the McFee orthogonal lead system (McFee and Parungao, 1961). The dogs were positioned in dorsal or right lateral recumbency; alligator-clip electrodes were saturated with 50% ethanol and attached to the skin. The ECGs were interpreted according to the criteria for ECG evidence of digoxin action previously reported (Fillmore and Detweiler, 1973). The configuration of the ECG in the Y lead of the McFee orthogonal system is comparable to Einthoven bipolar lead II; the McFee Z lead resembles somewhat VlO of the canine unipolar chest leads ; and the McFee X lead is recorded between a pair of electrodes on either side of the thorax, i.e., this lead is related to the canine Wilson system thoracic unipolar leads recorded from the right (CVSRL) and left (CV6LL, CV6LU) thorax (Detweiler 3 From the beagle colony of the Division of Veterinary Medical Research, Food and Drug Administration, Beltsville, Maryland.
SUBACUTE DIGOXIN TOXICOSIS IN DOGS
285
et al., 1968; Fillmore and Detweiler, 1973; McFee and Parungao, 1961). Thus the thoracic leads of the two systems, although related, are not identical. The signs of digoxin toxicosis previously classified by Fillmore and Detweiler (1973) as presumptive or reliable for the Wilson lead system were modified for the McFee system as follows. Presumptive signs.T-wave flattening or reversal : lead X, Z; S-T segment deviation, less than 0.15 mV; wandering pacemaker; variable P-R interval without dropped beats; P-R interval increased 20 % over control but less than 150 msec. Reliable signs. Sinus tachycardia greater than 200; sinus bradycardia less than 50; 1” atrioventricular block (P-R interval greater than 150 msec); incomplete atrioventricular block with dropped beats (second degree); atrioventricular dissociation; paroxysmal atria1 tachycardia (PAT) with or without block; ectopic atria1 beats; fusion beats ; interventricular block; S-T segment deviation greater than 0.15 mV; ventricular ectopic beats. The numerical scoring system for grading intensity of the ECG effects of digoxin, proposed earlier for the Wilson lead system (Fillmore and Detweiler, 1973), was used to quantify the ECG effects observed with the McFee lead system. This scoring system provides for quantification of ECG effects (a) on a daily basis (digoxin toxicosis score); (b) for the duration of the study period (total digoxin score) ; and (c) average effect over the study period (digoxin toxicosis index; DTI). The ECG scoring system, although arbitrary, was designed to reflect the degree of ECG abnormality observed in records taken on any given day. A score of 0 indicated that none of the ECG signs listed above was observed; 1 indicated one presumptive sign, and 2 indicated two or more presumptive signs. A score of 3, 4, or 5 indicated that 1, 2, or 3 reliable signs were observed, respectively. As in the previous work (Fillmore and Detweiler, 1973) with this scoring system, more than three reliable signs of digoxin toxicosis were not observed in any set of ECGs from a single dog. Hematology and Serum Chemistry Determinations
Blood samples for hematology and serum chemistry determinations were collected from 29 dogs (24 digoxin-treated and 5 control dogs) two to four times during the predose control period and at 1-3-day intervals during the treatment period. Additional baseline control values were tabulated from 168 normal beagle dogs in the same colony (Table 1). Complete blood counts and serum chemistry determinations were performed by an independent commercial laboratory. Serum chemistry determinations were performed by automated techniques and included total protein, albumin, uric acid, blood urea nitrogen (BUN), creatinine, glucose, sodium, potassium, chloride, calcium, inorganic phosphorus, alkaline phosphatase, serum glutamic-oxaloacetic transaminase (SGOT), lactic dehydrogenase (LDH), and creatinine phosphokinase (CPK). Serum digoxin concentrations of 20 dogs in the study were determined by radioimmunoassay; the blood samples for these determinations were collected at I-Zday intervals, in the morning, 24 hr after the previous dose. Necropsies
During the study, five dogs died from digoxin toxicity and a necropsy was performed within 12 hr of death. Four treated dogs and one control dog were killed by electro4 Schwarz/Mann,
Orangeburg, New York.
VALUES
0 Division
of Veterinary
Medical
Hematology Erythrocytes (x 106/mm3) Leukocytes (x 103/mm3) Hemoglobin (g/100 ml) Hematocrit (%) Mean corpuscular hemoglobin concentration, (%)
Research
76 76 77 23 23
Ekltsville,
34.6 k 2.2
43 Colony,
6.82 + 12.6 + 15.97 + 47.6 +
Maryland.
32-45
42
56 79 81 81
91 92 90 29 28
90 88 90 90 89 63 90 61 88
Number of
DETERMINATIONS
18-70 2.5-6.2 6.7-l 1.3 142-168 4.5-5.3
4.6-7.6 1.2-3.3 55-120 lo-24 0.34.9 0.6-l .o 20-177 37-260 25-300
Range
1
1.09 5.21-10.63 4.0 7.3-24.3 1.67 12.3-19.5 5.4 36-55
12.8 1.01 0.83 6.4 0.25
57 74 73 75
f. + + f. +
47.4 4.28 10.16 151.2 4.96
77 78 77 59 77 56 76
0.61 0.80 21.2 5.4 0.16 0.18 31.4 50.4 65.6
Mean *SD
TABLE HEMATOLOGY
Males
AND
6.47 + 1.90 rt 87.9 + 16.2 f 0.55 k 0.71 + 67.8 + 93.7 + 117.3 +
of dogs
CHEMISTRY
Beagle
FOR SERUM
Serum chemistry Total protein (g/100 ml) Albumin (g/100 ml) Glucose (mg/ 100 ml) Blood urea nitrogen (mg/lOO ml) Uric acid (mg/lOO ml) Creatinine (mg/lOO ml) Alkaline phosphatase (mu/ml) Creatinine phosphokinase (mu/ml) Lactic dehydrogenase (mu/ml) Serum glutamic-oxaloacetic transaminase (mu/ml) Inorganic phosphorus (mg/lOO ml) Calcium (mg/ 100 ml) Sodium (mequiv/liter) Potassium (mequiv/liter)
Determination
BASELINE
15.0 0.99 0.69 5.0 0.36
0.57 0.80 24.8 5.3 0.18 0.10 39.6 39.6 84.4
AND
25-108 2.4-6.2 9.4-11.5 136-161 4.1-5.3
4.8-7.7 1.4-3.7 40-140 l&32 0.4-1.1 0.5-1.0 30-270 30-400 25-370
Range
FEMALE
34.6 + 1.4
32-37
6.69 &- 0.97 4.93-10.40 11.2k4.3 5.5-28.0 16.07 + 1.67 12.0-19.0 37-62 48.0 f 4.9
45.3 + 4.23 f 10.35 + 151.3 + 4.80 f
6.40 f 2.07 + 91.8 + 17.1 f 0.52 + 0.69 2 74.6 f 85.6 f 124.5 k
Mean fSD
Females
IN MALE
85
113 153 154 156
167 168 167 52 51
168 166 167 168 166 122 167 117 164
Number of dogs
BEAGLES”
Mean *SD
1.03 4.1 1.61 5.2
13.6 1.00 0.77 5.6 0.32
34.6 f 1.8
6.76 + 11.9 f 16.16 f 47.8 f
46.3 + 4.26 + 10.27 + 151.3 + 4.87 f
6.43 + 0.59 1.99 + 0.80 90.0 If: 23.2 16.6 * 5.4 0.53 + 0.17 0.70 + 0.14 71.5 + 36.1 89.5 + 45.1 121.2 k 76.1
Total
z 2 ’
2 $j
E
SUBACUTE
DIGOXIN
TOXICOSIS
IN DOGS
287
cution, and the tissues were preserved in 10 % phosphate-buffered formalin. The remaining dogs were anesthetized with sodium pentobarbital, the thorax was opened, and the heart was retrogradely perfused via the aorta with isotonic phosphate-buffered saline followed by 2% phosphate-buffered glutaraldehyde at a pressure of approximately 100 Torr. Complete necropsies were performed on all animals and representative sections from all body organ systems were fixed in 10% phosphate-buffered formalin. Fixed tissues were taken from multiple preselected areas of the myocardium to include all major anatomic components of the heart and from other major organs of the body, embedded in paraffin, sectioned at 6-pm thickness, and stained with hematoxyhn and eosin. Heart sections were also stained with Gomori’s aldehyde fuchsin trichrome stain. Studies on 24 dogs were conducted at the Division of Veterinary Medical Research (DVR) laboratories, Beltsville, Maryland. Studies on the remaining 10 dogs were conducted at the Ohio State University, Columbus, Ohio. RESULTS
Physical Examination The digoxin-treated dogs generally showed signs of CNS depression with decreased activity; they also had a decreased food and water intake, and a mild loss of weight during the treatment period. Vomiting often occurred during the 1 or 2 days after the loading or modified-loading dose but did not persist. Diarrhea was not present in any dog. Dehydration was usually very mild; however, in one trial with four dogs, dehydration became severe during the period when the serum digoxin concentration was above 6 rig/ml but became less severe with decreased serum digoxin. The severity of these signs appeared to be well correlated with the digoxin concentration in serum. Representative samples of these changes in two dogs are illustrated in Fig. I. In all dogs, heart rate was determined from the ECG each day, and during the control period it ranged from 90 to 130 beats/min after the dogs became accustomed to having ECGs recorded. There was a sharp drop in heart rate to 60 % of control values (50-90 beats/min) by 6 hr following the loading dose. By 24-48 hr the heart rate had returned to control predose values. During the periods of most severe toxicity, the heart rate was elevated to 130-190 beats/min (Fig. 1). Body temperature was inversely related to the concentration of serum digoxin and other related signs of toxicity. The temperature was as much as 3°F lower than control predose temperature in the dogs most severely affected. Temperature decreased by approximately 1°F when serum digoxin concentrations exceeded 4 rig/ml and decreased further at more elevated concentrations. Serum Digoxin Serum digoxin concentrations varied considerably among individual animals. In eight dogs of the group given digoxin 5 days/week in which serum digoxin was determined, the concentration generally remained between 1.5 and 5.0 rig/ml by 4 days after the loading dose. After 2 days without treatment, however, the serum digoxin concentration was less than 1.2 rig/ml, and with the modified loading dose, gradually rose to 2.0-3.5 ng/rnl with 4 additional days of treatment. The same pattern held for two dogs that were treated for a third week following a second 2-day period with no dose; the concentrations finally reached 2.5 and 4.0 rig/ml on the seventeenth day of the study.
288
TESKE
ETAL.
Dogs treated 7 days/week with digoxin generally had higher digoxin concentrations in serum than those treated 5 days/week; these concentrations were sustained unless the dose was reduced. The difficulty in establishing a stable toxic serum concentration of digoxin is illustrated in Figs. 2 and 3. The dogs whose concentrations are shown in Fig. 2 received digoxin at 25 pg/kg on Day 1 following the loading dose on Day 0 and maintained very high toxic concentrations of digoxin, between 8 and 10 rig/ml, until the DIGOXW TOXICITY IN THE DOG 89V
59x
IO -
- I25
Ge-
‘98
4-
0:- g E-
c t 4’
600400-
6
4
IO
I2
I4
I6
lb
i
6
600,
bFs3
4
;a *5 Q-
IO
1
- 1000
200-
.
C
F? az E’
2
4
6
6
IO
I2
I4
I6
C
I8
160
102
100
wo 101 ,
2
I
4
I
6
I
6
1
0
1
I
I
IO
I2
I4
I6
I6
‘60 I60 120 zi 30
C
2
4
6
6
IO
I2
I4
I6
16
600 400 200
t :w
C
2
4
6
6
W
3
.
C
ze 85
I25
‘8
2
4
6
6
IO
12
I4
I6
I6
i[?K C 600 400-l
2
4
1
E 6
6
IO
-3 fE zg
rm
k400
2oo~200
DAY9 FIG. 1. Correlation of toxicosis indices with serum digoxin concentration administered for two separate dogs. Digoxin was given 7 days/week.
and dose of digoxin
dose was reduced to 10 pg/kg or omitted entirely during the second week of treatment. Although the dogs in this trial (Fig. 2) responded very similarly, four dogs in a subsequent trial (Fig. 3) had marked variations in their response to iv digoxin administration. In this second trial, no dose was given on Day 1 following the loading dose, and in two of the dogs (59X and 64X, Fig. 3), there was a slow gradual increase of serum digoxin within the toxic range above 2.5 rig/ml while the dose was maintained relatively constant at 25-35 pg/kg. In dog 63X, serum digoxin rapidly reached severely toxic levels, above 6 rig/ml, at the 25-,ug/kg dose level. In contrast, dog 61X had very low concentrations of serum digoxin which only reached mildly toxic levels after 8 days of treatment in spite of a dose increase to 35 ,ug/kg.
SUBACUTE
DIGOXIN
SERUM
MGOXIN
TOXICOSIS AND DIGOXIN
289
IN DOGS DOSE
FIG. 2. Serum digoxin concentrations are indicated for four separate dogs by the connected symbols and the total dose administered iv to each dog by the nonconnected symbols. Injections were started on Day 0 and were given 7 days/week. Serum digoxin concentrations for each day were determined on samples collected prior to administration of digoxin.
ll?zcil Ik/ M SERUM
DIGOXIN
AND DBOXH
DOSE
64X
59x
lo 6 6 4 2 0
126
IO
126
loo
6
loo
76
6
75
60
4
60
26
2
25
0
0
2
4
6
6
IO
024660
63X
IO
126
IO
I25
loo
6
100
I6
6
76
4
SO
4
60
2
2s
2
26
6 6
0
0 0
2
4
6
6
IO
0
2
4
6
6
IO
DAYS FIG.
3. Serum digoxin concentration
and digoxin dose for four separate dogs treated 7 days/week.
Electrocardiograms
Many alterations in ECGs were encountered during the course of the experiment. The most frequently found changes included first and second degree heart block (prolonged P-R interval with or without dropped beats), ventricular extrasystoles or
0 1 0 0 0 0 0 0 0 0 0 -0 -0 -
0 0 1 0
Digoxin 5 days/ week 21U (Control) 80T (Control) 12U (Control) 81T (Control)
31T 38U 86s 79T 31u 42U 74T 82T 58V 60V 94u 61V 62V 63V
0
Dog number
DAILY
0
1 3 0 4 3 1 1 3 0 2 1
0 0 0 0
1
DIG~XIN
2 3 1 0 0 2 1 0 3 3 2 1 2 3
0 0 0 0
2
1 3 3 0 0 1 0 1 2 3 3 3 3 3
0 0 0 0
3
TOXICITY
2 1 1 1 1 0 3 3 3 1 2 3 4 1
0 0 0 0
4
SCORES
-
-
-
-
-
-
-
-
-
6
-
5
IN BEAGLE
TABLE
2
13 3 3 3 0 2 3 FD 3 1 1 0 3 3
0 0 10 0
7
3 2 1 3 0 4 3
3 1 2 3 3 3
2 4 4 4
0 0 11 0
9
3 3 4 11 12 3
0
0 0
8
day
2 3 3 3 4 3
3 4 4 5 11--l 3 3
0
0 0
10
Experiment
--2 FD --3
--3 --0
3 3 s s s 0 4 3
-----0 ---
---0
13
DIGOXIN
--0 -----0
12
WITH
3 4 3 FD
0 0 l--l 0
11
DOGS FOLLOWING TREATMENT TOXIC DOSAGE LEVELS’
14
4
4
3
3 3 3
3 3 3
4
5 4 4
0 0 1 0
16
3 4 4
0 0 0 0
15
ADMINISTERED
17
3
3
3 3 3
4 4 FD
AT
3
2
3 3 3
5 4
18
2.69 3.14 2.72 2.62 1.50 2.14 2.28 1.75 2.25 1.87 2.12 2.08 3.38 3.00
DTI
1.35
-3.66
4 3 4
:
3 4 4 4
-
2.00
4 4 4 4 3 3
-
2.53
3 4 4 4 3 3
3 3 3 1 1 3
0
2.53
4 4 4 4 3 4
3 3 3 3 1 3
--
3.15
4 4
3 4
3 5 2 3 1 3
2.66
-
5 4
FD
__-
-
55 44
-3 -4 -3 -3
3.57
3
1.50
3.29
5 4
4 3 3 3 11
-
3.50
3.14
5 4
3 4 2 3
-
3.62
2.83
5 -
3 3 2 4 0
-
3.28
2.60
3 3 1 3 3
0
3.28
2.64 3.35 2.00 1.78 1.14 1.44 3.25 3.33 3.64 3.38 2.50 3.12 2.11 2.55
0 0
2.39
gx
$j z
%
,,,,,
,,,
,,
1.35
2.00
2.00
3.00 2.66
2.75
2.91
,”
3.25
I,,
3.20
/I,
? g 2
4
3 2 4 ---
----
0 0 3 3 2 3
---
3 3
0
1.86
’ The digoxin toxicity scores are a subjective numerical scoring system of presumptive (l-2) and reliable (3--S) signs of toxicity (see Methods for the criteria for electrocardiographic evidence of digoxin toxicity). DTI, digoxin toxicosis index; FD, found dead; S, sacrificed.
1.28
0 3 4 3 3
3 3
0
1.86
8w
0
Mean
0 3 lll-10 110 0 3 3 3 0 112 14 lll-113
0
1.64
2.58
00 14 02 01 02 00 04 03 02 00 00 00 00 00
-0 o------
0
36 36T 68T 98U 99u 97u 45w 54w 89V 9ov 59x 63X 61X 64X
Mean Digoxin 7 days/ week 94V (Control) 96V (Control)
292
TESKE ET AL. DIGOXIN
TOXICITY (45W) McFEE
IN THE LEAD
DOG
2
lrec CONTROL
I DAY
FIG. 4. McFee lead Z electrocardiogram during control period and 1 day after the loading dose. Ventricular extrasystoles are present on the Day 1 recording. oIoox#
TOXICITY IN THE DDG (59X) McFEE LEAD Y
CONTROL
SERUM DIGOXM 80 rig/ml
2 DAYS
SERUM DlOOXlN * 3.0 rig/d
7 DAYS
SERUM DIGOXIN = 6.4 rig/ml
SERUM IO DAYS 5. McFee lead Y electrocardiogram during week. At 7 and 10 days there is second degree heart ~asuperimposed on the preceding T-wave, but are FIG.
DlGOXlN=lO rig/ml subacute digoxin toxicity in a dog treated 7 days/ block with dropped beats. On Day 10 the P-waves clearly identified on the beat following a dropped
SUBACUTE
DIGOXIN DEOXW
7 CMYS SERUM
293
IN DOGS
TOXlClTY IN THE DOG (SW McFEE LEAD 2
IO DAYS SERUM
DlG4D.Otqhl
17 DAYS
TOXICOSIS
SERUM
DIG= 10.0 rig/ml
DlG=5.1 rig/ml
FIG. 6. McFee lead 2 electrocardiogram during subacute digoxin toxicity in a dog treated 7 days/ week. There is bradycardia at 6 hr, S-T segment depression at 7 days, second degree heart block with dropped beats at 10 days, and paroxysmal A-V dissociation with sinus block and ventricular extrasystoles at Day 17.
d LL
III*
LL
IImv
1
Irr
L.
. 4% I45
I70
220
250
FIG. 7. McFee lead X, Y, and Z electrocardiograms from a dog (59X) that developed signs of severe toxicity during the course of digoxin treatment 7 days/week. One-millivolt standards are marked for each lead; X2 = 1 mV is twice the amplitude marked by the bar. Paper speed was identical (30 mm/set) for all tracings. Changes in the wave form of the QRS complex and T-wave (ventricular aberration) are apparent with increasing serum level of digoxin and prolongation of the P-R interval.
escape beats, deviation of the S-T segment, and alterations in the QRS and T-wave form. The daily digoxin toxicity score based on the finding of presumptive or reliable ECG signs of toxicity and the DTI for each dog are listed in Table 2. Ventricular ectopic
294
TESKE ET AL. DIGOXIN DAY SERUM
DIG
C (0)
6h (7.0)
I (4.6)
TOXICITY IN THE DOG (61X1
2 (I.8 i)
3 (1.3)
4 (I.
7 2.0)
8 (2.9)
9 (3.2)
IO 14.~
4J
II
1
m
-fI T
I II5
I II5
120
120
120
125
160
FIG. 8. McFee lead X, Y, and Z electrocardiograms from a dog (61X) treated 7 days/week and having only mild to moderate digoxin toxicosis. Wave form changes are less pronounced than those in Fig. 7.
beats, when present, usually occurred during the first week of treatment and resulted in a digoxin toxicosis score of 3 or 4 (Fig. 4). The P-R intervals became progressively prolonged in nearly all dogs, with dropped beats occurring during the second week of treatment in many dogs (Fig. 5). Other less common arrhythmias sometimes found in instances of severe toxicity (Fig. 6) are listed in Methods (Electrocardiography). Deviations of the S-T segment were occasionally found but seldom were greater than 0.25 mV. Changes in the wave form pattern of the QRS complex and the T-wave (ventricular aberration) were frequent findings (Figs. 7 and 8). Hematology and Clinical Chemistry
Total erythrocyte count, hemoglobin, packed cell volume, red cell indices, and total and differential leukocyte counts remained within normal limits throughout the study, except for four dogs (45W, 54W, 89V, and 90V) that became severely dehydrated with resulting hemoconcentration associated with severely toxic concentrations of digoxin in serum. Mild fluctuations in packed cell volume seen occasionally in other dogs were associated with the state of hydration but were not outside normal limits. Alterations in serum CPK and LDH activities and in BUN, creatinine, sodium, chloride, and potassium occurred in dogs with digoxin toxicosis. All other clinical chemistry determinations remained within normal limits. Tables 3 and 4 list the range of values for those determinations with altered values, together with the median for each group and the number of determinations that exceeded normal limits for each experimental day. The median is given since the curve is not normally distributed but skewed to the right. Normal baseline values for dogs in the beagle colony from which the dogs in this study were derived are given in Table 1. Representative examples of the day-to-day fluctuations of the various indicators of digoxin toxicity are shown jn Fig. 1 BUN values were elevated to over 25 mg/lOO ml in 21 of 28 dogs examined, including all of the dogs treated 7 days/week. Serum creatinine was elevated up to 8.1 mg/lOO ml
-
(60)
40-280 (75) 30-130 (75) 30-145 (90) 3@230 (110) 30-120 (50) 30-380 (50) 40-350
(80)
40-100 (70) 50-150
Range
CPK (mu/ml)
215
215
O/5
219
o/9
o/9
l/10
o/10
o/10
Xln
OF SUBACUTE
LDH (mu/ml>
40-1005 (105) 45-780 (90)
(210)
100-700 (390) 105-660 (350) 80-810 (405) SO-450
(210)
95-500 (143) 651220 (155) 55-670
215
2/5
215
6/9
5/9
6/9
2/10
O/l 0
o/10
X/n
TOXICITY
Range
DIGOXIN
TABLE DOSE)
3
23-46 (44
(36)
17-54
(23
10-17 (15) 1O-28 (17) 14-55 (19) 12-25
15-100 (20)
(21)
10-18 (15) 18-28
Range
415
415
O/5
t/9
t/9
O/9
2/10
l/10
o/10
X/n
BUN (mg/lOO ml)
(~-DAYS/WEEK
CHEMISTRY
(0.8)
0.7-1.0 (0.7) 0.6-1.0 (0.8) 0.7-l .2
(0.6)
Oh-I.0
(0.6)
0.5-l .o
(0.6)
0.660.9 (0.7) 0.7-0.9 (0.8) 0.6-5.5 (0.7) 0.6-1.0
Range
l/5
o/5
o/5
o/9
o/9
O/9
l/10
o/10
O/10
Xln
Creatinine (mg/lflO ml)
ON SERUM
DOGS’
126-158 (141)
(136)
140-151 (146) 14&150 (146) 144153 (149) 130-153 (145) 130-150 (1W 129-149
(146)
146-158 (151) 141-152
Range
215
315
2/5
2/9
o/9
o/9
o/9
O/l0
O/10
X/n
Sodium (mequiv/liter)
OF BEAGLE
4.4-5.1 (4.8) 3.3-5.3 (4.7) 4.1-4.8 (4.5) 3.8-5.3 (4.4) 4.2-5.3 (4.9)
(4.6)
4.4-5.2 (4.8) 3.6-5.3 (4.7) 4.4-5.2 (4.9) 4.3-5.5
Range
O/5
215
l/5
219
o/9
l/9
O/9
l/10
O/10
X/n
Potassium (mequiv/liter)
’ CPK, creatinine phosphokinase; LDH, lactic dehydrogenase; BUN, blood urea nitrogen; X/n, number of values which exceed 95 % confidence limits of normal values/number of observations for that date. Normal values are derived from Table 1 and are: CPK < 185 mu/ml, LDH < 275 mU/ml, BUN < 27 mg/lOO ml; sodium 140-160 mequiv/liter; potassium 4.2-5.4 mequiv/liter. Median values are given in parentheses.
18
16
14
11
9
7
4
2
0
Day
EFFECT
318
w3)
97-305
(160)
91-280
076)
70-508 (180) 120-585 (170) 10&490
(165)
6/ 10
l/9
3/12
6/l 3
5/ 13
70-l 187 7/14 (170) 40-1070 6/14
(185)
o/14
40-171 (140) 90-230 (115) 90-417
3/10
X/n
Range
(210)
35-475 (185) 80-445 (190) 41-700 (147) 55-600 (145) 56-820 (131) 58-550 (150) 52-700
(136)
lW450 (130) 104-180
(160)
75-215
Range
LDH (mUhI)
S/10
219
2112
6/l 3
3/l 3
5114
2114
O/8
2/10
O/14
X/n
15-120 (37)
(32)
12-100 (40) 15-191 (4-v 15-87
(20)
13-165 (225) 13-165
(2%
9-24 (14) lo-21 (13) 9-82 (30) 14-202
Range
6/10
519
6/12
9/13
6/13
6/14
7/14
418
o/10
o/14
Xln
BUN (mg/lOO ml)
(0.8)
O-6-2.6 (1.0) 0.5-1.6 (1.0) 0.6-2.4 (1.1) 0.7-3.2
W3)
0.5-2.1
(0.8)
0.6-5.6 (1.1) 0.7-8.1
(0.8)
0.6-0.9 (0.7) 0.6-1.1 (0.7) 0.4-2.8
Range
3/10
519
6/12
6/l 3
5/12
6/14
8/14
3/8
l/10
O/14
X/n
Creatinine (mg/lOO ml)
123-159 (135) 119-175 (132)
(128)
121-152 (142) 48-150 (139) 39-l 50 (145) 114-147 (140) 116142
(148)
149-153 (150) 146151 (149) 142-152
Range
7/10
6/9
6/11
8/13
4/l 1
4/8
4/11
O/S
O/8
o/14
X/n
Sodium (mequiv/liter)
3.8-5.5 (5.0) 3.47.7 (5.1) 3.9-6.8 (4.8) 4.0-5.6 (4.9) 3.7-5.2 (4.7) 3.3-5.8 (4.3) 3.2-5.7 (4.3)
(4.6)
4.6-5.2 (4.9) 4.3-5.2 (5.0) 4.4-5.0
Range -.___
8/10
519
2/l 1
3/l 3
3/13
4/l 1
2/l I
O/8
O/8
O/14
X/i1
Potassium (mequiv/liter)
4 DOSE) ON SERUM CHEMISTRY OF BEAGLE DOGS"
-
61, 82
(56)
71-l 10 (75) 32-97
19,106
107-l 12 (110) 99-l 06 (100) 64/106 (87) 20-l 10 (59) -
Range
212
717
314
l/2
517
718
O/8
X/11
Chloride (mequiv/liter)
n CPK, creatinine phosphokinase; LDH, lactic dehydrogenase: BUN, blood urea nitrogen; X/n, number of values which exceed 95 s,; confidence limits ot normal values/number of observations for that date. Norma1 values are derived from Table 1 and are: CPK < 185 mu/ml, LDH 4 275 mu/ml, BUN < 27 mg/lOO ml; sodium 140-160 mequiv/liter; potassium 4.2-5.4 mequiv/liter; chloride 100-I 14 mequiv/liter. Median values are given in parentheses.
18
15
12
10
8
Day
CPK (mu/ml)
TABLE EFFECT OF SUBACUTE DIGOXIN TOXICIIY(~-DAYS/WEEK
Q k .
;;t g
E
SUBACUTE
DIGOXIN
TOXICOSIS
297
IN DOGS
in parallel with the increased BUN. In dogs with the most severely elevated BUN (over 100 mg/lOO ml), there was frequently a mild increase in packed cell volume associated with decreased water intake and dehydration. Serum sodium and chloride were moderately to severely reduced in several dogs concomitant with the elevated BUN and were especially marked in those dogs that had the most severe dehydration. Serum potassium was mildly elevated in several dogs, although, in a few, serum potassium was lower than normal (Tables 3 and 4). Serum CPK and/or LDH activities were mildly to moderately elevated at some time during treatment in 7 of the 10 dogs examined that were on the 5-day/week dose and in 11 of 14 dogs on the 7-day/week dose. In many dogs the increased activity of serum enzymes conincided with the highest serum digoxin concentrations and other signs of toxicity, but this was not always the case. In some dogs there appeared to be no obvious correlation of elevated serum enzyme activity with other signs of toxicity; CPK and LDH activities were usually both elevated at the same time, but sometimes one was elevated while the other remained within normal limits. Because isoenzymes of CPK or LDH were not determined, it is not possible from the clinical chemistry data to identify which body organ was injured to release them. However, the only organs with lesions found at necropsy were the heart and kidneys (vide i&z). Table 5 summarizes the changes that occurred with digoxin administration. TABLE 5 SUMMARY OF CHANGES OCCURRING WITH DIGOXIN ADMINISTRATION CORRELATED WITH THE DEGREE OF TOXICOSIS AND SERUM DIGOXIN CONCENTRATIONS
Nontoxic or mild toxicity
Moderate toxicity
Severe toxicity
Sodium (mequiv/liter) Chloride (mequiv/liter) Potassium(mequiv/liter) CPK, LDH
K2.5 Normal Normal Normal Normal
