Rapid administration of high-dose human antibody fab fragments to dogs: Pharmacokinetics and toxicity

FUNDAMENTALANDAPPLIEDTOXICOLOGY

17,83-91

(1991)

Rapid Administration of High-Dose Human Antibody Fab Fragments to Dogs: Pharmacokinetics and Toxicity’

DANIEL

E. KEYLER,*,~

DAVID M. SALERNO,$ MARYANN M. MURAKAMI,$ GEORGE RUTH,§ AND PAUL R. PENTEL*‘*

*Department oj‘Medicine, Division of Clinical Pharmacology and Toxicology, University of Minnesota Medical School, Hennepin County Medical Center, Minneapolis, Minnesota 55415: *Department ofMedicine, Division of Cardiology. University of Minnesota Medical School, Hennepin County Medical Center, Minneapolis, Minnesota 55415; TCollege of Pharmacy, University of Minnesota. Minneapolis, Minnesota 55455; §College qf Veterinary Medicine. University gf Minnesota. St. Paul. Minnesota 55455

Received September Il. 1990; accepted January 29. 1991

Rapid Administration of High-Dose Human Antibody Fab Fragments to Dogs: Pharmacokinetics and Toxicity. KEYLER, D. E., SALERNO, D. M., MURAKAMI, M. M.. RUTH, G.. AND PENTEL, P. R. (199 1). Fundam. Appl. Toxicol. 17, 83-9 1. The treatment of drug overdose with drug-specific antibody fragments may require very high antibody doses. To address the feasibility of this therapy, we studied the pharmacokinetics and toxicity of high-dose human nonspecific Fab fragments in beagles. Three dogs received 5.3 g/kg Fab iv over 1 hr. Because nephrotoxicity was observed, three subsequent dogs received 3.2 g/kg. The fraction of the Fab dose excreted in urine (IO f 6%) was lower than reported values for either high or low doxs of Fab in other species. The terminal serum elimination half-life (42 hr for the higher and 48 hr for the lower dose) was also longer than reported values for other species, due to lower renal and nonrenal Fab clearance. Fab administration was tolerated without adverse hemodynamic effects.One of three dogs at each dose developed transient oliguria. All dogs developed a transient but marked increase in the serum creatinine concentration. At 2 weeks creatinine clearance had returned to normal. Urinary protein and albumin excretion at 2 weeks were within the normal range for dogs but were increased over their baseline values. The histology of all organs was normal at 3 weeks by light microscopy, and renal histology by electron microscopy was also normal. The mechanism of Fab nephrotoxicity, not observed previously with high-dose Fab in rats or lower doses of Fab in other species including dogs, is not clear. These data suggest that further study of the potential toxicity of high-dose Fab, and its reversibility, is needed to assessthe feasibility of treating drug overdose with this antibody fragment. The long terminal half-life of high-dose Fab in the dog and its low renal clearance contrast with values observed with lower doses of Fab in other species but would not be expected to preclude the use of high-dose Fab for drug overdose. o 199I society of Toxicology.

The most effective treatment for massive digoxin overdose is drug-specific antibody Fab fragments. Administration of Fab at a dose that is equimolar to the body burden of dir Supported by PHS Grant MH42799. z To whom correspondence should be addressed at Department of Medicine, Hennepin County Medical Center, 70 I Park Avenue, Minneapolis, MN 554 15.

goxin rapidly and completely reverses toxicity. Despite its efficacy, the clinical use of drugspecific antibodies has not yet been extended to other drugs (Colbum, 1980; Scherrmann et al., 1989). Digoxin is an unusually potent toxicant (Wenger, 1985). Most drugs have lethal doses 2 to 3 orders of magnitude higher and would presumably require correspondingly 83

0272-059019 I $3.00 Copyright 0 I99 I by the Society of Toxicology. All rights of reproduction in any form reserved.

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higher doses of Fab to treat an overdose. The need to administer such large doses of Fab (e.g., 5.3 g/kg Fab would be equimolar in terms of binding sites to a potentially lethal 35 mg/ kg tricychc antidepressant overdose) raises questions regarding both their pharmacokinetics and their potential toxicity. To address these questions, we previously administered 7.5 g/kg of nonspecific human Fab to rats iv over 1 hr (Pentel et al., 1988). No adverse pathophysiologic effects were noted. Of particular importance because of the potential for antibody-induced immunologic injury to the kidney, creatinine clearance, urinary protein excretion, and renal histology were not affected by Fab administration. The disposition of high-dose Fab was remarkably similar to that of much lower doses (0.0 1-O. 1 g/kg) of digoxin-specific Fab (prepared from sheep antiserum) in dogs (Lloyd and Smith, 1978) and humans (Schaumann, 1986), with a terminal serum half-life of 16.3 hr and 32% renal excretion. These data, then, support the feasibility of using Fab for treating overdoses due to drugs that have a high toxic dose. The current study extends these observations with the administration of high-dose Fab to dogs. Compared to rats, transient renal insufficiency was observed, urinary excretion of Fab was lower, and the terminal serum half-life was longer. METHODS Animal preparation. Because of the cost of preparing large amounts of Fab, small male beagles (7.3 to I 1.4 kg) were used and the study was limited to six animals. Dogs were housed in metabolic cages with ad libitum access to standard dry dog food and water. A 24-hr urine collection and a midpoint serum sample were obtained prior to Fab administration for creatinine clearance and urinary protein determinations. On the day of Fab administration dogs received no food or water after midnight. Dogs were anesthetized between 7 and IO AM with pentobarbital 30 mg/ kg iv and ventilated via an endotracheal tube with 100% oxygen at a tidal volume of 20 ml/kg and a rate of 14 ventilations/min. Both femoral veins, the right femoral artery, and a brachial vein were cannulated. The right atrium was cannulated via the right femoral vein for central venous pressure measurement, with catheter position confirmed by fluoroscopy. Catheters were kept patent with

ET AL. 5% glucose at a rate of I ml/mitt. Arterial blood gaseswere obtained just prior to Fab infusion and the ventilator adjusted to maintain an arterial blood pH of 7.35-7.45. Fab was infused via the left femoral vein cannula and venous blood was obtained from the left brachial vein cannula. Blood pressure was monitored using a pressure transducer and polygraph. The electrocardiogram was monitored continuously and recorded every 5 min. Protocol. Fab was administered via the left femoral vein over 1 hr. The first three animals received 5.3 g/kg, with this dose chosen to approximate a Fab dose equimolar to a commonly fatal (35 mg/kg) tricyclic antidepressant overdose. Because all three animals developed transient renal insufficiency, the next three animals received a lower dose, 3.2 g/kg Fab (chosen to be equimolar to a minimal lethal dose in humans). Venous blood (3 ml) was obtained just prior to Fab infusion and at intervals after the start of Fab infusion (Fig. 1). Blood samples were immediately centrifuged and serum was stored at -70°C until assayed for Fab concentration. Blood pressure, heart rate, right atria1 pressure, and the electrocardiogram were monitored for 1 hr after Fab infusion: catheters were removed; and animals were then returned to their cages. The total amount of iv fluid administered during anesthesia ranged from 280 to 300 ml. All urine was collected for 8 days (but only 5 days for animal I owing to an initial underestimation of Fab half-life) and stored at -70°C until assayed for Fab concentration. Twenty-four-hour urine collections were obtained at 2 weeks and animals were euthanized at 3 weeks by iv pentobarbital administration. Tissue samples were immediately frozen at -20°C or fixed in formalin or glutaraldehyde for histologic examination. Tissues collected were heart, brain, liver, kidney. quadriceps muscle. spleen, and lung. Assays. Creatinine was measured by the method of Ja& (Jaffe, 1896). Protein was measured by dye binding (Bradford, 1976). Fab concentrations were measured by radial immunodiffusion (Pentel et a/.. 1988) with a sensitivity of 0.1 &ml. Control dog serum did not cross-react with Fab. Creatinine kinase activity was measured by immunoelectrophoresis (Vaidya. 1986). Urinary albumin concentration was determined by nephelometry with rabbit anti-dog albumin (Organon-Teknika, Westchester, PA) that does not cross-react with human Fab (Malkus, 1978). The fractional excretion of sodium was calculated as the urinary sodium clearance divided by the urinary creatinine clearance. Urine heme pigment was estimated using a semiquantitative peroxidase-sensitive dipstick (Multistix SC, Miles Inc., Elkhart. IN) that does not distinguish between hemoglobin and myoglobin. Preparation ofFab. Fab fragments were prepared from lyophilized human IgG (Sandoglobulin, gift of Sandoz Pharmaceuticals, East Hanover, NJ) by papain digestion and purified by protein A chromatography as previously described (Pentel et al., 1988). Contaminants present in this Fab preparation are not detected by the radial immunodiffusion assayused to measure Fab concentrations (Pentel et al., 1988).

HIGH-DOSE

Fab PHARMACOKINETICS

Fab was concentrated to 0.3 g/ml for administration in phosphate buffered saline, pH 7.4, using an Amicon 8400 with 30 kDa cutoff membrane. Each dose of Fab was assayedfor protein and Fab concentration, and homogeneity was analyzed by SDS-PAGE and densitometry. Doses were based upon Fab concentration measured by radial immunodiffusion, using commercially available human Fab that was homogeneous by SDS-PAGE as a standard. Immunoelectrophoresis of serum from dogs treated with Fab (Pentel et al., 1988) was performed to determine whether immunoreactive metabolites of Fab were formed in vivo. Hisrology. Formalin-lixed tissueswere examined by light microscopy using hematoxylin-eosin and periodic acidSchiffstains. Kidney was examined by electron microscopy of glutaraldehyde-fixed tissue, postfixed in osmium tetroxide and stained with uranyl acetate and lead citrate. Estimation of pharmacokinetic parameters. Estimates of 11, B, OL,and 0, t/2a and t/2p corrected for infusion time, were obtained by fitting a biexponential equation to the concentration time data using the computer program PC NONLIN version 3.0, 1989 (Statistical Consultants Inc., Lexington, KY). The steady state volume of distribution (V,), volume of the central compartment (V,), and total body clearance (Cl,) were determined by model-independent methods (Gibaldi and Perrier, 1982). Cumulative urinary excretion of Fab after Day 8 was calculated by extrapolating the area under the excretion-time curve. Because fl for urinary excretion could not be accurately estimated for animal 1. Fab excretion for this animal was not extrapolated. Statistical evaluation. Mean values for cardiovascular (Table 2) and urinary parameters (Table 4) were compared using one-way ANOVA. The paired, two-tailed t test was used for all other comparisons.

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j I,,,,,,,, 12345678

Days FIG. 1. Serum Fab concentration after administration of Fab 5.3 g/kg (n = 3) and 3.2 g/kg (n = 3) over 1 hr in dogs. Solid lines are computer-fitted estimates using a twocompartment model, bars represent standard deviations.

2 and 5 are similar using either a two- or a three-compartment model (42 vs 53 hr, and 48 vs 67 hr). For uniformity, the two-compartment values for all animals are used in Table 1. Cumulative urinary Fab excretion is shown in Fig. 2. Collections were lost for animal 6 on Days 5 and 6. Toxicity. Baseline values for all parameters shown in Tables 2-4 were within the normal range for dogs (Osborne and Stevens, 1981). Fab infusion produced no changes in mean arterial blood pressure, heart rate, or the electrocardiographic QRS interval (Table 2). Right atria1 pressure increased but remained in the RESULTS normal range of 2-8 mm Hg for anesthetized dogs (Ettinger, 1989). Arterial blood pH after Fab assay. Fab homogeneity determined by Fab infusion was also unchanged (range 7.36 SDS-PAGE was 48%. Immunoelectrophoresis to 7.43). All animals awoke from anesthesia of urine and serum from treated animals re- within 6 hr and behaved normally for the first vealed a single precipitin arc that comigrated 24 hr. Animals 2 and 5 appeared listless after with freshly prepared Fab. awakening with poor intake of food and water Pharmacokinetics. Mean serum Fab con- and decreased movement in the cage. Behavior centrations and computer-fit curves are shown returned to normal by Day 3. Transient oliin Fig. 1 for each Fab dose. Fab was detectable guria was observed in two of the six animals in serum for up to 10 days. Estimates of phar- (Fig. 3), and polyuria was observed in all anmacokinetic parameters are shown in Table 1 imals. A transient increase in the serum crefor each dose separately and for both doses atinine concentration was observed in all ancombined. The serum Fab concentrations of imals, including those without oliguria (baseanimals 2 and 5 are best described by a three- line serum creatinine concentration 7.0 + 0.6 compartment model; all others are well de- &ml, peak 48 & 2 pg/ml, p < 0.0 1). The peak scribed by a two-compartment model. The serum creatinine concentration occurred at terminal Fab elimination half-lives for animals Days l-3. At 2 weeks the serum creatinine

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TABLE 1 ESTIMATED PHARMACOKINETIC PARAMETERS mg/ml Fab dose 5.3 g/kg

3.2

g/kg

All animals

hrr’

liter/kg

Dog

A

B

a

P

1

28.3 26.2 28.9

3.20 4.63 14.32

0.106 0.106 0.300

0.011

0.19

0.54

2 3

0.016 0.023

0.17 0.12

Mean

27.8

7.38

0.171

0.017

4 5 6

31.6 19.3 18.1

4.53 1.61 2.09

0.484 0.049 0.214

Mean

23.0

2.74

Mean +SD

a

u

v,

vss

ml/kg/hr

hr

ClB

Ch

a t/2

P t/2

Fun,

0.39 0.28

9.78 9.74 7.48

1.29 4.29 1.32

6.6 6.5 2.3

59.7 44.6 29.7

0.06 0.12 0.06

0.16

0.40

9.00

2.30

4.0*

41.9*

0.08

0.019 0.014 0.011

0.08 0.16 0.16

0.42 0.22 0.76

10.35 6.25

0.61

11.51

2.55

1.4 14.0 3.2

37.3 51.1 64.3

0.05 0.09 0.21

0.250

0.014

0.13

0.46

9.37

1.53

2.7*

48.4'

0.12

0.210 0.162

0.016 0.005

0.14 0.04

0.43 0.19

9.19 1.95

1.92 1.32

3.3h

44.56

0.10 0.06

1.44

Note. ‘4, B. 01,and p are computer-estimated values corrected for infusion time. V, central volume of distribution, Vss steady-state volume of distribution, Clr, total body clearance. Cla renal clearance, Furine fraction of dose excreted

in urine. ’ Mean values for A and B are not calculated because these parameters are dose dependent. * Harmonic mean.

concentration and creatinine clearance had returned to baseline for all animals. Urinary total protein and albumin excretion at 2 weeks were within the normal range for dogs (Osborne and Stevens, 198 1) but increased over the baseline value (Table 3). Urine specific gravity, sodium concentration, and osmolality were lower at 24 hr than at baseline but did not differ from baseline at 14 days (Table 4). Trace or small amounts of heme pigment were present in the urine of all animals at 24 hr. The mean serum creatine kinase concentrations were 173 + 65 IU at baseline, 1080 2 327 IU at 11 hr, and 1201 f 588 IU at 24 hr (p < 0.0 1 for both values compared to baseline). Organ histology. All tissues appeared normal by light microscopy and renal histology was also normal by electron microscopy. There were no basement membrane deposits suggestive of immune complex deposition.

ilarly prepared high-dose Fab in rats in both pharmacokinetics and toxicity. The terminal half-life of Fab for both doses in dogs was

24 -

.E 5

123456789 Days

DISCUSSION High-dose

Fab infusion

in beagle dogs dif-

fered from previously reported results for sim-

FIG. 2. Cumulative urinary Fab excretion. Collections for dog 6 on Days 5 and 6 were lost (indicated by the dotted tine). The mean cumulative Fab excretion was 10 f 6% of the dose.

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Fab PHARMACOKINETICS

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TABLE 2 EFFECTOF FAB ADMINISTRATION ON CARDIOVASCULARFUNCTIONS Mean arterial pressure (mm Hg) Preinfusion Midinfusion Postinfusion

149 f 17

152 + 17 148 _+ 14

Right atrial pressure (mm Hg)

Heart rate (min-‘)

QRS duration (msec)

1.1 f 1.4 4.9 + 1.6 4.2 t- 1.4

170+- 19 178 + 17 169 + 20

38.8 zk 5.3 38.9 + 5.2 38.8 + 5.3

(b) (b)

’ Data for all six animals combined (mean + SD). * p < 0.01 compared to preinfusion value.

longer than that of rats (44.5 vs 16.3 hr) and urinary excretion of Fab was lower in dogs (10 vs 3 1%) due to lower renal and nonrenal

clearance (Pentel et al., 1988). In dogs, Fab infusion was well tolerated acutely but was followed by transient renal insufficiency. Al-

Fab 5.3 g/kg

Fab 3.2 glkg

60

600

0

2

4

6

8

10

12

14

0

2

4

6

8

10

12

14

Days FIG. 3. Serum creatinine concentration and urine output for individual dogs. Left panels-Fab 5.3 g/kg, right panels-Fab 3.2 g/kg. Day 0 represents baseline (pre-Fab) values. All dogs had transient increases in the serum creatinine concentration and urine output. Two dogs (3 and 4) also had transient oliguria. Dog I CO), 2 Cd,3 (a), 4 (0). 5 (A) and 6 (0).

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ET AL.

TABLE 3 EFFECTS OF FAB ON RENAL FUNCTION

Creatinine clearance (ml/min/kg) Fab dose 5.3 g/kg

3.2g/kg All animals

AT 14 DAYS

Urinary protein excretion (mg/kg/24 hr)

Urinary albumin excretion bw/kg/24 W

Dog

Baseline

14 days

Baseline

14 days

Baseline

14 days

I 2 3 4 5 6 Mean SD

1.39 2.52 2.44 3.71 4.20 2.18 2.14 1.03

1.08 2.50 2.53 3.18 2.90 2.34 2.42 (NS) 0.73

2.3 8.8 6.4 2.8 5.6 6.4 5.4 2.5

10.0 47.6 26.3 5.1 15.2 20.0 20.7* 15.1

0.3 0.6 0.5 0.4 0.8 0.5 0.5 0.2

I.0 2.9 1.5 1.0 0.9 1.3 1.4* 0.8

Note. NS = p > 0.05. * p < 0.01 for 14 days vs baseline.

though creatinine clearance and urine output apparently longer terminal half-life of highhad returned to normal by 2 weeks, incomdose Fab in the current study is a result of plete resolution of toxicity over the study pe- being able to measure serum Fab concentrariod was suggested by increased urinary pro- tions for longer than 48 hr. In our previous tein excretion. study of high-dose Fab in rats, however, serum The pharmacokinetics of the 5.3 and 3.2 g/ Fab concentrations were measured for 80 hr, kg doses of Fab were similar and will be dis- and considering only the first 48 hr would not cussed together. Aside from the previous study have changed the terminal serum half-life of of rats (Pentel et al., 1988), we are aware of Fab (Pentel et al., 1988). Thus there does appear to be a difference in the terminal serum no pharmacokinetic data regarding comparably high doses of Fab. For lower (tracer) doses half-life of high-dose Fab in the rat and of nonspecific Fab administered to rats, ter- the dog. minal serum half-lives of 1.7 hr (Arend and With regard to the potential therapeutic use Silverblatt, 1975) and 3.6 hr (Wochner et al., of high-dose Fab, half-life per se does not ap1967) have been reported. These very low val- pear to influence efficacy; digoxin-specific Fab ues were probably due to measuring serum and IgG are both effective in reversing digoxin Fab concentrations for only 6 and 10 hr, re- toxicity despite the much longer half-life of spectively, so that the estimated half-lives re- IgG (Lloyd and Smith, 1978; Smith et al., flected distribution as well as elimination. 1979; Ochs and Smith, 1977). Recurrence of Longer terminal half-lives have been reported toxicity due to release of IgG-bound digoxin for digoxin-specific Fab in dogs ( 17.1 hr, Lloyd does not occur. A potentially longer terminal and Smith, 1978), baboons (9.1 hr, Smith et half-life for high-dose Fab would not therefore be expected to compromise its efficacy. Simal., 1979) and humans (25.4 hr, Schaumann, 1986). In each case, the terminal half-life was ilarly, the smaller fraction of the Fab dose excalculated using serum Fab concentrations creted in urine in dogs would not be expected measured for only 48 hr. In the current study, to compromise therapeutic efficacy. Intact IgG omitting Fab concentrations obtained after 48 is effective in reversing digitoxin toxicity dehr would have resulted in a mean terminal spite its very low renal excretion compared to half-life of only 20.3 hr for the high dose and Fab (5 vs 25% of the administered dose over 16.9 hr for the low dose. It is possible that the 96 hr in rhesus monkeys) (Ochs and Smith,

HIGH-DOSE

Fab PHARMACOKINETICS

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89

1977). The pharmacokinetics of high-dose Fab in dogs is, therefore, compatible with therapeutic efficacy for drug overdose. The potential nephrotoxicity of high-dose Fab is, however, of some concern. Urinary total protein and albumin excretion at 2 weeks, although within the normal range for beagle dogs (Osborne and Stevens, 1981) were greater than their baseline values. The increased total protein excretion could have been in part due to urinary Fab, as the Fab assay used will not detect concentrations < 1 mg/ml. However, the urinary albumin assay used does not detect human Fab and the urinary albumin concentration measurement would not be similarly affected. In contrast, no toxicity was noted after an even higher dose of Fab in rats (Pentel et al., 1988). No toxicity has been noted after administration of lower doses of digoxin-specific Fab (0.02-o. 1 g/kg) in any species, except for a low incidence of allergic reactions in humans (Wenger et al., 1985). Specifically, nephrotoxicity has not been reported in dogs after digoxin-specific Fab, although detailed renal function studies were not reported (Ochs and Smith, 1977). The mechanism of renal Fab toxicity in the current study is unclear. Fab infusion was tolerated acutely with no hemodynamic changes. Central venous pressure was normal prior to Fab and did not change during infusion. It is unlikely that animals became dehydrated during Fab administration as they received 80 to 190 ml of Fab in phosphate-buffered saline, additional 5% glucose, and central venous pressure did not change. Poor intake of food and water was probably a result of azotemia rather than its cause, because the onset of azotemia was already evident by 24 hr. Rhabdomyolysis was considered as a cause of renal insufficiency because of the rapidity with which the serum creatinine concentration increased. However, the increase in serum creatine kinase concentration observed was smaller than that usually associated with myoglobinuric renal failure in humans (Grossman et al., 1974). Thus sufficient rhabdomyolysis to cause renal failure is unlikely

90

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but cannot be excluded. A serum sickness re- 3 weeks and the lack of any structural abnoraction to the infused Fab or contaminant malities are compatible with a resolving and could cause renal injury, but this typically oc- reversible lesion. Some reversible toxicity may curs l-2 weeks after provocation (Dixon and be acceptable when drug-specific Fab is used Wilson, 1979). Examination of renal tissue by to treat an immediately life-threatening drug light and electron microscopy, which was nor- overdose, so that the use of Fab for this purmal at 3 weeks, does not comment on the pose might not be precluded by transient renal mechanism of the observed renal insult. insufficiency. Recent data regarding antibody Nephrotoxicity could have been due to the treatment of tricyclic antibody toxicity in rats Fab per se, or to contaminants in this prepa- also suggest that substantial reversal of toxicity ration. We did not attempt to purify the Fab can be achieved using antibody doses lower further because of its lack of toxicity in rats. than those administered in the current study In addition, the required quantities of Fab are (Pentel et al., 1991). The current findings, costly to prepare and further purification de- however, emphasize the need to further charcreases the Fab yield. While it is unlikely that acterize Fab renal toxicity with regard to dose, impurities in this preparation affected Fab dosing rate, species of origin of the Fab, and pharmacokinetics, a more homogeneous Fab impurities in the preparation. They also sugpreparation is clearly needed to clarify the role gest that the use of antibody fragments other of impurities in the observed nephrotoxicity. than Fab may be of interest. If toxicity was due to Fab per se, it could be caused by intact Fab or a metabolite. This ACKNOWLEDGMENTS study was not designed to characterize Fab We thank Dts. C. Osborne, B. Anderson, T. O’Brien, metabolism, other than to show that any meand J. Crosson for assistance with histologic studies, Drs. tabolites formed did not interfere with the imP. Abraham and W. Elmquist for helpful discussions, and munodifhtsion assay for Fab. It is possible that M. Madsen for technical assistance. toxicity was due to the human origin of the Fab leading to immunologic injury such as REFERENCES immune complex deposition in the dog, although similarly prepared Fab produced no AREND, W. P., AND SILVERBLATT. F. J. (1975). Serum disappearance and catabolism of homologous immurenal toxicity in rats (Pentel et al., 1988). While noglobulin fragments in rats. Clin. Exp. Immunol. 22. a control group was not included in this study, 502-5 13. it is unlikely that pentobarbital anesthesia was BRADFORD, M. (1976). A rapid and sensitive method for the cause of renal insufficiency. This anesthetic the quantitation of microgram quantities of protein utiis widely used for dogs and impairment of lizing the principle of protein-dye binding. .4nal. Biochem. 72,248. renal function has not been reported (Soma, COLBURN, W. A. (1980). Specific antibodies and Fab frag197 1). Intact (undigested) human IgG has not ments to alter the pharmacokinetics and reverse the been reported to impair renal function in papharmacologic/toxicologic effectsof drugs. Drug Metab. tients, but it has only been administered at Rev. 11, 223-262. lower doses (up to 500 mg/kg) and at lower DIXON, F. S., AND WILSON, C. B. (1979). Immunological renal injury produced by formation and deposition of rates than in the current study (Stiehm et al., immune complexes. In Immunologic Mechanisms of 1987; Ochs et al., 1980). Renal Disease (C. B. Wilson, Ed.), pp. 4-6. Churchill The reversibility of Fab renal toxicity is of Livingston, New York. considerable importance for its potential clinETTINGER, S. J. (1989). Textbook of Veterinary Internal ical use. At 2 weeks creatinine clearance had Medicine. p. 933. Saunders, Philadelphia. returned to normal but urinary protein excre- GIBALDI, M., AND PERRIER,D. (1982). Phurmucokinetics. 2nd ed, pp. 67-69. Dekker, New York. tion was modestly increased. Whether toxicity GROSSMAN, R. A., HAMILTON, R. W., MORSE, B. M., would have completely resolved over a longer PENN. A. S., AND GOLDBERG, M. (1974). Nontraumatic period requires further study. However, the rhabdomyolysis and acute renal failure. N. Engl. J. Med. 291,807~8 I 1. mild nature of the functional abnormalities at

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JAFFE,M. Z. (I 896). Physiol. Chem. 10, 39 1. LLOYD, B. L., AND SMITH, T. W. (1978). Contrasting rates of reversal of digoxin toxicity by digoxin-specific IgG and Fab fragments. Circulation f&,280-283. MALKUS, H., BACHBAUM, P., AND CASTRO, A. (1978). An automated turbidimetric rate method for immunoglobulins. Clin. Chem. Acta 88, 523-530. OCHS, H. D., BUCKLEY, R. H.. PIROFSKY, B., FISHER, S. H., ROUSSELL, R., ANDERSON, C. S., AND WEDCWOOD, R. J. ( 1980). Safety and patient acceptability of intravenous immune globulin in 10% maltose. Lancer 2, 1158-l 159. OCHS, H. R., AND SMITH, T. W. (1977). Reversal of advanced digitoxin toxicity and modification of pharmacokinetics by specific antibodies and Fab fragments. J. Clin. Irwest. 60, 1303-1314. OSBORNE,C. A., AND STEVENS,J. B. (1981). Handbook q/‘Canine and Feline Urinal&. Ralston Purina Co., St. Louis. PENTEL, P. R.. KEYLER, D. E., GILBERTSON.D. G., RUTH, G.. AND POND, S. M. (1988). Pharmacokinetics and toxicity of high doses of antibody Fab fragments in rats. Drug Metab. Dispos. 16, 14 1- 145. PENTEL. P. R., BRUNN, G. J.. POND, S. M., KEYLER, D. E. ( 199 I). Pretreatment with drug-specific antibody reduces desipramine cardiotoxicity in rats. Li/e Sci. 48, 675683.

SCHAUMANN. W., KAUNANN. B., NEUBERT, P.. AND SMOLARZ. A. ( 1986). Kinetics of the Fab fragments of

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diogoxin antibodies and of bound diogoxin antibodies in patients with severedigoxin intoxication. Eur. J. Clin. Pharmacol. 30,527-533. SCHERRMANN, J. M., TERRIEN, N., URTIZBEREA, M., PIERSON, P., DENIS, H., AND BOURRE, J. M. (1989). Immunotoxicotherapy: Present status and future trends. J. Toxicol. Clin. Toxicol. 27, 1-35. SMITH. T. W., LLOYD, B. L., SPICER,N., AND HABER, E. (1979). Immunogenicity and kinetics of distribution and elimination of sheep digoxin-specific IgG and Fab fragments in the rabbit and baboon. Clin. Exp. Immunol. 36,384-396.

SOMA, L. R. (197 1). Veterinary ilnesthesia. (L. R. Soma, Ed.). p. 284. Williams & Wilkins, Baltimore. STIEHM, E.. ASHIDA, E., KIM, K. S., WINSTON, D. J., HAAS, A., AND GALE, R. P. (1987). Intravenous immune globulins as therapeutic agents.Ann. Int. Med. 107,367382.

VAIDYA, H. C., MAYNARD, Y., DIETZLER, D. N., AND LADENSON, J. H. (1986). Direct measurement of CK MB activity in serum. Clin. Chem. 32,657-663. WENGER, T. L., BUTLER. V. P.. HABER, E., AND SMITH, T. W. (1985). Treatment of 63 severely digitalis-toxic patients with digoxin-specific antibody fragments. J. Am. Coil. Cardiol. 5, 118A-123A. WOCHNER. R. D., STROBER.W.. AND WALDMAN, T. A. (1967). The role of the kidney in the catabolism of Bence-Jones proteins and immunoglobulin fragments. J. Exp. Med. 126, 207-221.