JPET Fast Forward. Published on February 24, 2004 as DOI: 10.1124/jpet.104.065250 JPET FastThis Forward.article has not Published been copyedited on and February formatted. The24, final 2004 version as DOI:10.1124/jpet.104.065250may differ from this version.

JPET #65250 1

Characterization of the Neutralizing Activity of

Digoxin-specific Fab

toward Ouabain-like Steroids Downloaded from

Mark A. Pullen, David P Brooks and Richard M. Edwards jpet.aspetjournals.org

Department of Renal Biology,

GlaxoSmithKline Pharmaceuticals at ASPET Journals on September 26, 2021 King of Prussia PA 19406

Copyright 2004 by the American Society for Pharmacology and Experimental Therapeutics. JPET Fast Forward. Published on February 24, 2004 as DOI: 10.1124/jpet.104.065250 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #65250 2

a) Running Title: Characterization of Digoxin-specific Fab b) Mark A. Pullen

709 Swedeland Road, Mail Code UW2521

King of Prussia, PA 19406

Phone (610)-270-4988

Fax (610)-270-5681 Downloaded from [email protected] c) Text: 23 pages

Tables: 2 jpet.aspetjournals.org

Figures: 5

References: 40 at ASPET Journals on September 26, 2021 Abstract: 174 words

Introduction: 575 words

Discussion: 1615 words d) Nonstandard abbreviations: EDLF-endogenous digoxin-like factor. e) Recommended section assignment: Cardiovascular JPET Fast Forward. Published on February 24, 2004 as DOI: 10.1124/jpet.104.065250 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #65250 3

ABSTRACT

Digoxin-specific Fab (Digibind) is a mixture of anti-digoxin Fab fragments prepared from sheep sera and is used as a treatment for digoxin poisoning. Digoxin-specific Fab has been shown to neutralize an endogenous

Na/K ATPase inhibitor (EDLF) in rats and humans and to lower blood pressure.

Although the exact structure of EDLF is unknown, compounds identical to or Downloaded from structurally related to ouabain, bufalin and marinobufagenin have been detected in mammalian plasma. In this study some structural characteristics of EDLF were jpet.aspetjournals.org inferred from the ability of digoxin-specific Fab to neutralize the Na/K ATPase inhibitory activity of several known cardenolides and bufodienolides. Additional structural information was obtained from 3H-ouabain binding and ELISA at ASPET Journals on September 26, 2021 experiments. Digoxin-specific Fab had the ability to interact to some extent with all of the cardenolides and bufodienolides tested. However, digoxin-specific Fab was more than 20-fold more potent in neutralizing ouabain and bufalin than marinobufagenin. The antihypertensive effect of digoxin-specific Fab seen in preeclampsia and animal models of hypertension may therefore be due to a molecule identical to or structurally similar to ouabain or bufalin.

JPET Fast Forward. Published on February 24, 2004 as DOI: 10.1124/jpet.104.065250 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #65250 4

The existence of an endogenous digoxin-like Na/K ATPase regulatory factor (EDLF) or factors have been known for some time and has been reviewed elsewhere (Schoner, 2002). They are thought to play a major role in the cardiovascular system ( Ke, 2001) in part by affecting the contractility of heart and vascular smooth muscle tissue by elevating intracellular calcium (Levy et al., Downloaded from 1995). The exact structure(s) of EDLF is unknown but there have been reports that it is closely related to or identical to the cardenolide, ouabain which is produced by adrenal cells in culture (El-Masri et al., 2002) and is found jpet.aspetjournals.org circulating in human plasma (Hamlyn et al., 1991). This ouabain-like substance has also been found in brain tissue (Huang and Leenen, 1996). Additional studies at ASPET Journals on September 26, 2021 have reported that marinobufagenin, a bufodienolide found in toads, is present in human plasma and is elevated in congestive heart failure (Fridman et al., 2002), and in Dahl salt-sensitive hypertensive rats (Fedorva et al., 2001). An elevation of both a ouabain-like and marinobufogenin-like EDLF has been measured in plasma from patients with preeclampsia (Lopatin et al., 1999) and in volume expanded anesthetized dogs (Bagrov et al., 1996). A factor that cross-reactives with a monoclonal antibody against bufalin, another bufodienolide, has been found in human and rat plasma (Oda et al., 2001). In addition, an EDLF structurally related to bufalin has been found in human placenta (Hilton et al.,

1996). Rabbit antisera with specificity against the bufodienolide proscillardin A reacts with a substance elevated in the plasma of hypertensive humans (Sich et al.,

1996) Some digoxin-like substances cross-react with anti-digoxin antibodies and JPET Fast Forward. Published on February 24, 2004 as DOI: 10.1124/jpet.104.065250 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #65250 5

are sometimes measured using digoxin assays (Morris et al., 1990; Namoi et al.,

1991; Dasgupta and Emerson, 1998).

Digoxin-specific Fab (Digibind ) is a mixture of Fab fragment produced by papain digestion of antibodies raised in sheep against a digoxin-human albumin conjugate (Curd et al. 1971). The Fab fragments are purified by affinity Downloaded from chromatograph. The main therapeutic indication for this product is digoxin overdose. The utility of this agent has also been studied in the neutralization of jpet.aspetjournals.org similar cardiotoxins from plants and toad venom (Dasgupta and Emerson, 1998;

Brubacher et al., 1999). The ability of digoxin-specific Fab to interact with an endogenous EDLF has also been measured in animal studies of corticotropin- at ASPET Journals on September 26, 2021 induced hypertension (Li et al., 1997) and 5/6 nephrectomized hypertensive rats

(Kaide et al., 1999). Digoxin-specific Fab has been shown to neutralize the effect of ouabain (Krep et al., 1995) and EDLF (Balzan et al., 1991) in vitro. Both digoxin-specific Fab and intact anti-digoxin or anti-ouabain antibodies have been shown to have hemodynamic and cardiac effects (Ke, 2001).

EDLF has been shown to increase significantly during pregnancy-induced hypertension and preeclampsia (Adair et al., 1996; Goodlin, 1988; Lopatin et al.,

1999). Digoxin-specific Fab lowered blood pressure and increased urine output in preeclamptics (Adair et al., 1996; Goodlin, 1988) suggesting a role for EDLF in the pathogenesis of preeclampsia.

JPET Fast Forward. Published on February 24, 2004 as DOI: 10.1124/jpet.104.065250 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #65250 6

Since the actual structure of EDLFs remains controversial and elusive, a systematic study of the ability of digoxin-specific Fab to neutralize the Na/K

ATPase inhibitory activity of 15 different digoxin-like molecules in vitro was conducted. The purpose of this study was to gain knowledge of the structure- activity relationship concerning the ability of digoxin-specific Fab to neutralize the functional activity of these molecules in inhibiting Na/KATPase. Additional Downloaded from structure-activity information was obtained by cross-reactivity ELISA experiments using digoxin-specific Fab as the primary antibody and competition jpet.aspetjournals.org experiments of 3H-ouabain binding to digoxin-specific Fab.

at ASPET Journals on September 26, 2021 JPET Fast Forward. Published on February 24, 2004 as DOI: 10.1124/jpet.104.065250 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #65250 7

METHODS

All Na/K ATPase inhibitors, ammonium molybdate, polyvinyl alcohol and malachite green were obtained from Sigma (St. Louis, MO). Human heart membranes were from Biological Services Inc. (Wilmington, DE). 3H-Ouabain was from Perkin Elmer Life Sciences (Boston, MA). Immunosorb 96 well plates from Nunc (Rochester, NY). Digibind  was from GlaxoSmithKline (Research Downloaded from

Triangle Park, NC). All experiments were performed with a single lot, 2C3451.

jpet.aspetjournals.org Na/K ATPase assay:

ATPase activity was measured by detecting the inorganic phosphate produced from the hydrolysis of ATP by a reaction with malachite green and at ASPET Journals on September 26, 2021 ammonium molybdate under acidic conditions in the presence of polyvinylalcohol. Briefly, 0.4–0.7 µg of human heart membrane protein was added to 50 µl of HEPES buffer pH 7.5 consisting of 3 mM ATP, 4mM MgCl,

144 mM NaCl, 5 mM KCl and 0.1 mM EDTA. Because potassium shifts Na/K

ATPase to a conformation that does not bind ouabain (Lingrel and Kuntzweiler

1994), potassium was added last.

After 1-hour incubation at 37°C with shaking, 200 µl of color reagent was added. Color reagent was made 1 hour prior to use and consisted of 0.8 mM malachite green base, 8.0 mM ammonium molybdate tetrahydrate, 1N HCl and 4 mg/ml polyvinylalcohol. Polyvinylalcohol was dissolved in boiling water and ammonium molybdate tetrahydrate was dissolved in 6N HCl at 6 times their final JPET Fast Forward. Published on February 24, 2004 as DOI: 10.1124/jpet.104.065250 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #65250 8

concentration. Assays were conducted in 96 well plates, which were read in a

Molecular Devices (Sunnyvale, CA) Thermomax spectrophotometer at 650 nm after 4 minutes.

Human cardiac membranes were chosen for these studies because of the sensitivity of cadiac tissue to cardioglycosides and ouabain in vivo, while rat Downloaded from ATPase, particularly in kidney, is relatively resistant to ouabain. Initial experiments with the human cardiac membrane showed almost no ouabain sensitivity, so the membranes were treated with sodium dodecyl sulfate buffered jpet.aspetjournals.org with bovine serum albumin (BSA) as described previously (Forbush, 1983). The ideal conditions for treating the cardiac membranes involved the addition of at ASPET Journals on September 26, 2021 membrane protein and SDS at the same concentration of 0.36 mg/ml in 50 mM

HEPES pH 7.5. Following 10 minutes of SDS treatment at room temperature, the mixture was diluted 6 fold with 0.3 % BSA in 50 mM HEPES. The ATPase activity was about 80% ouabain sensitive and stable upon freezing. Ouabain sensitivity of the cardiac membrane was maintained when the final SDS concentration in the ATPase assay was 0.01 mg/ml.

Neutralization of Cardioactive Steroids:

The ability of digoxin-specific Fab to neutralize the inhibition of ATPase activity of several cardenolides and bufodienolides was measured by its ability to cause a rightward shift in the inhibition curves of these compounds. Ten µl of digoxin-specific Fab diluted in 50 mM HEPES 0.3 % BSA were incubated for 1 JPET Fast Forward. Published on February 24, 2004 as DOI: 10.1124/jpet.104.065250 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #65250 9

hour 37°C with 10 µl of increasing concentrations of cardenolide or bufodienolide. Twenty µl of cardiac membranes with sodium, magnesium,

EDTA and ATP were added five minutes prior to adding potassium. The assay was stopped 1 hr later with color reagent and read at 650 nm after 4 minutes. IC50 values were determined using GraphPad Prism software. Schild analysis was

used to calculate the concentration of digoxin-specific Fab needed to shift the Downloaded from cardenolide inhibition curve two-fold to the right. Because the ATPase enzyme, which competes with the digoxin-specific Fab for binding to the inhibitor, was jpet.aspetjournals.org held constant, these values are not true pA2 values. The values reflect the concentration of digoxin-specific Fab needed to neutralize the ATPase inhibitors at their IC50 concentration at ASPET Journals on September 26, 2021

ELISA Assay Using digoxin-specific Fab as the Primary Antibody:

Nunc Immunosorb plates were coated with dixogin-BSA conjugate by adding 200 µl of conjugate at a concentration of 1 µg/ml in 0.1M sodium carbonate/bicarbonate buffer pH 9.5 to each well. Plates were stored at 4°C for up to one week. Plates were blocked with 1% BSA in phosphate buffered saline

(PBS) for 1 hour prior to the assay. Plates were washed 3 times with PBS with

0.02 % Tween 20. Fifty µl of PBS-0.2% BSA followed by 50 µl of increasing concentrations of cardioactive steroids were added to the plates. One hundred µl of 0.5 µg/ml digoxin-specific Fab in PBS-0.1% BSA was added to each well.

After 2-hour incubation with shaking at 37°C, the mixture was removed from the JPET Fast Forward. Published on February 24, 2004 as DOI: 10.1124/jpet.104.065250 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #65250 10

plate and the plate was washed three times with PBS-0.02% Tween 20. Digoxin- specific Fab that bound to the digoxin-BSA conjugate was detected by adding 200 ul peroxidase labeled donkey anti-sheep antibody diluted 1:5000 in PBS-0.1%

BSA to each well and incubating for an additional hour at 37°C. The plates were then washed three times with PBS-0.02% Tween 20 to remove unbound anti- sheep antibody. Two hundred µl of peroxidase substrate, OPD-fast from Sigma, Downloaded from was added to each well and color was allowed to develop for 12 minutes. Plates were read at 450 nm in Thermomax (Molecular Devices, Sunnyvale, CA) jpet.aspetjournals.org spectrophotometer. Preliminary experiments determined the saturating concentration of digoxin-specific Fab and donkey antibody dilution to produce an absorbance of 1.0 at 14 minutes. Background was measured with 10 µM digoxin at ASPET Journals on September 26, 2021 and was less than 10% of total.

3H-Oubain binding to digoxin-specific Fab

Digoxin-specific Fab was coated onto Packard (Perkin Elmer Life

Sciences, Boston, MA) 96 well Optiplates at a concentration of 2 µg/ml in 200 µl of 0.1M sodium carbonate/sodium bicarbonate buffer pH 9.5 at least over night.

Plates were blocked with 1.0% BSA in PBS for one hour. Plates were then washed three times with PBS-0.02% Tween-20 prior to the addition of 100 µl

PBS 0.15% BSA. Fifty µl of several concentrations of ATPase inhibitors were added to each well immediately before the addition of 50 µl of 6.4 nM 3H- ouabain (666 GBq/mmol) diluted in PBS 0.1% BSA. After 1-hour incubation at JPET Fast Forward. Published on February 24, 2004 as DOI: 10.1124/jpet.104.065250 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #65250 11

37°C, unbound 3H-ouabain was removed by washing the plate three times with

PBS-0.02% Tween-20. Two hundred µl of Packard Microscint 20 scintillation cocktail was added to each well and plates were counted in a Packard Top Count

NXT (Perkin Elmer Life Sciences, Boston, MA).

Statistical Analysis Downloaded from

All IC50 values and apparent pA2 values presented were calculated using

GraphPad Prism  software from data of experiments performed at least three jpet.aspetjournals.org times. Statistical analysis of IC50 values was performed by two-tailed t-test of log IC50. at ASPET Journals on September 26, 2021 JPET Fast Forward. Published on February 24, 2004 as DOI: 10.1124/jpet.104.065250 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #65250 12

RESULTS

The general steroid structure of the cardenolides and bufodienolides used in these experiments is shown in Figure 1. Cardenolides have a 5-membered lactone ring attached to the steroid nucleus at position 17 while the bufodienolides have a six-membered lactone ring. All cardenolides have a hydroxyl at the 14 Downloaded from position while some bufodienolides have a 14-15 epoxide. The steroid nucleus is sometimes glycosylated in the 3 position. The steroid substitutions are summarized in Table 1. jpet.aspetjournals.org

Of the various cardenolides and bufodienolides used in this study, at ASPET Journals on September 26, 2021 neriifolin, convallatoxin and ouabain were the most potent inhibitors of human cardiac Na/K ATPase (Table 2). In general, glycosylation had little effect on the potency of cardenolides without a 5-hydroxyl group. Digoxin was not statistically different from digoxigenin while the difference between digitoxin and digitoxigenin was significant (p<.005) but small (Fig 2A, Table 2). In contrast, cardenolides with a 5 hydroxyl and a sugar group such as convallatoxin and ouabain were at least 10-fold more potent than their corresponding aglycones, strophantidin and ouabagenin respectively (p<.005)(Fig. 2B, Table 2).

Digoxin-specific Fab was found to shift the inhibition curves of these compounds to the right when preincubated with the cardenolide or bufodienolide prior to Na/K ATPase assay (Table 2). For example, concentrations of digoxin- JPET Fast Forward. Published on February 24, 2004 as DOI: 10.1124/jpet.104.065250 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #65250 13

specific Fab ranging from 100 nM to 2500 nM shifted the marinobufagenin curve several folds (Fig. 3A). The apparent pA2 values for digoxin-specific Fab against this bufodienolide were calculated by Schild plots (Fig. 3B). The slopes of the

Schild plots for all of the compounds were not significantly different from –1.0

(average +/- SEM n=16 –1.1 +/- 0.05) and linear (R2 = 0.955 +/- 0.01) indicating

that the inhibitory effect of digoxin-specific Fab toward these compounds was Downloaded from competitive. Digoxin-specific Fab was able to neutralize all of the inhibitors to some extent (Table 2). jpet.aspetjournals.org

Since the neutralization of ouabain by digoxin-specific Fab was observed, experiments were conducted to study the ability of the cardenolides and at ASPET Journals on September 26, 2021 bufodienolides to compete with 3H-ouabain for binding to digoxin-specific Fab.

Preliminary experiments showed that 3H-ouabain bound with a reasonably high affinity of 0.8 nM (data not shown). Because digoxin-specific Fab is a mixture of many different Fabs from a large population of sheep, binding studies with 3H- ouabain should give information about the subpopulation of antibodies that have a high affinity for ouabain. Both digoxin and convallatoxin, which are not hydroxylated in the eleven position, competed better for 3H-ouabain binding than oubain itself, which is hydroxylated in the eleven position (p<.005) (Fig. 4). This would indicate that while the binding sites of certain antibodies can recognize ouabain, they recognize the antigen, digoxin, better. The rhamnoside form of JPET Fast Forward. Published on February 24, 2004 as DOI: 10.1124/jpet.104.065250 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #65250 14

strophanthidin, convallatoxin, was more potent than the aglycone (p<.005).

Bufodienolides with 14-15 epoxides were the least potent competitors (Table 2).

ELISA experiments were performed to determine the structural characteristics of cardenolides and bufodienolides that can compete against digoxin-specific Fab binding to digoxin BSA conjugate (Table 2). Digoxin and Downloaded from digitoxin, which differ by one hydroxyl group in the steroid 12 position, had no statistical difference in potency (Fig. 5, Table 2). Convallatoxin, which has more differences in the number and position of hydroxyl groups in the steroid nucleus, jpet.aspetjournals.org loses binding ability. The importance of the glycoside in antibody interaction is again observed with the decreased binding ability of strophanthidin relative to at ASPET Journals on September 26, 2021 convallatoxin (p<.005). Marinobufagenin was a rather poor competitor (Fig. 5,

Table 2). JPET Fast Forward. Published on February 24, 2004 as DOI: 10.1124/jpet.104.065250 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #65250 15

DISCUSSION

The existence of an EDLF(s) involved in blood pressure control has been known for many years (reviewed in Schoner, 2002). While the exact chemical structure of this factor(s) is still debated, current evidence suggests that it is closely related to or identical to ouabain (Hamlyn et al., 1991), marinobufagenin

(Bagrov et al., 1998a), proscillardin A (Sich et al., 1996) or bufalin (Hilton et al., Downloaded from 1997). Digoxin-specific Fab has been shown to neutralize the effects of exogenous ouabain on central nervous system mediated changes in blood pressure

(Teruya et al., 1997), Rb uptake in erythrocytes (Balzan et al., 1991) and vascular jpet.aspetjournals.org smooth muscle contraction (Krep et al., 1995). Additional studies have shown that digoxin-specific Fab has the ability to neutralize an endogenous factor with at ASPET Journals on September 26, 2021 Na/K ATPase inhibitory activity similar to ouabain produced in human peritoneal dialysate (Tao et al., 1996; Krep et al., 1995), cord blood of neonates (Balzan et al., 1991.), and rat brain (Huang and Leenen, 1996). Digoxin-specific Fab has also been shown to reduce blood pressure in a rat corticotropin (Li et al., 1997) and 5/6 nephrectomy models (Kaide et al., 1999). Finally, digoxin-specific Fab has activity against endogenous Na/K ATPase inhibitors involved in hypertension associated with pregnancy. A substance that cross-reacts with digoxin immunoassays has been reported in patients with peeclampsia and pregnancy- induced hypertension (Kaminski and Rechberger, 1991; and Graves et al., 1995).

This has been further characterized as both an ouabain-like and marinobufogenin- like factor (Lopatin et al., 1999). There have been two reported clinical cases of JPET Fast Forward. Published on February 24, 2004 as DOI: 10.1124/jpet.104.065250 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #65250 16

digoxin-specific Fab treatment of preeclampsia (Adair et al., 1996) and eclampsia

(Goodlin, 1988).

A key unanswered question is the identity of the molecule(s) that digoxin- specific Fab neutralizes in vivo. In the present study we evaluated the ability of digoxin-specific Fab to neutralize the activity of several cardenolides and Downloaded from bufodienolides to provide additional information as to the structurally identity of

EDLF. Interestingly, digoxin-specific Fab demonstrated the ability to neutralize cardenolides that are glycosylated in the 3 position (digitoxin, neriifolin, jpet.aspetjournals.org convallatoxin and ouabain) with a similar potency compared to its ability to neutralize digoxin regardless of substitutions in the steroid portion of the at ASPET Journals on September 26, 2021 molecule. Not only could digoxin-specific Fab neutralize glycosylated inhibitors, but also their respective aglycones. However, the effect of digoxin-specific Fab could only be observed when the ATPase inhibitor concentration was close to its

IC50 value in the cardiac membrane system being studied. Higher concentrations of aglycones were used in the neutralization assays because they displayed lower potency than the glycosylated forms as characterized previously (Melero et al.,

2000). In virtually every case, when the inhibitor was present at its IC50 concentration, an equal concentration of digoxin-specific Fab was effective.

Digoxin-specific Fab can neutralize the activity of a variety of inhibitors. Because the neutralizing affinity of digoxin-specific Fab toward the inhibitors could not be observed below the IC50 value of the inhibitor in this system, additional studies JPET Fast Forward. Published on February 24, 2004 as DOI: 10.1124/jpet.104.065250 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #65250 17

were conducted to elucidate the interaction of digoxin-specific Fab with the inhibitors.

Binding studies with 3H-ouabain and ELISA experiments showed a decrease in potency with greater changes in the steroid nucleus. The aglycones digitoxigenin, strophanthidin and ouabagenin differ only in the number and Downloaded from position of hydroxyl groups and lose potency as they diverge in structure from digoxigenin. Rhamnosides of 5 hydroxy cardenolides are more potent than their jpet.aspetjournals.org respective aglycones in both assays. These data show that there are a population of Fab fragments in the mixture of Fabs that recognize several different positions on the steroid nucleus in addition to the glycosylated 3 position. This is in at ASPET Journals on September 26, 2021 contrast to the neutralization data with digoxin-specific Fab that demonstrated little difference between the steroid substitutions of inhibitors glycosylated in the

3 position.

Bufodienolides behave in a similar fashion to the cardenolides in some respects. The least substituted, bufalin, has the same structure as digitoxigenin except for the six membered lactone ring in the 17 position. This does not negatively effect Na/K ATPase activity. Bufalin is also neutralized by digoxin- specific Fab to a greater degree than would be expected based on its aglycone structure. Some data exists for endogenous bufalin-like Na/K ATPase inhibitor that reacts with bufalin specific antibodies and is elevated in the serum of salt sensitive Dahl rats (Oda M 2001). A bufalin-like molecule lacking a 14 hydroxyl JPET Fast Forward. Published on February 24, 2004 as DOI: 10.1124/jpet.104.065250 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #65250 18

and an unsaturated bond in the lactone ring have been identified in human placenta (Hilton et al., 1997). This molecule when prepared from androsterone has the ability to inhibit Na/K ATPase. Our results suggest that digoxin-specific

Fab may have some neutralizing activity toward these types of molecules.

Some of the bufodienolides have 14-15 epoxides instead of a 14 hydroxyl Downloaded from found in all of the cardenolides tested. This, along with acetylation of the 16 position, seems to have additional negative effects on digoxin-specific Fab's jpet.aspetjournals.org ability to neutralize these compounds and their potency in 3H-ouabain and ELISA assays. These positions do not appear to be as important as the steroid 3 position that is the site of glycosylation on both the cardenolides and bufodienolides. This at ASPET Journals on September 26, 2021 observation supports the lack of efficacy of digoxin-specific Fab in a mouse model of toad venom poisoning (Brubacher et al., 1999) and in clinical settings involving voluntary ingestion of toad venom extract (Brubacher et al., 1996).

Digoxin-specific Fab was only 55% effective in preventing death and 30% effective in preventing seizures in mice. When digoxin-specific Fab was used clinically to treat toad venom poisoning, one patient died and two recovered after receiving up to 20 vials of digoxin-specific Fab. Clearly, digoxin-specific Fab is less effective in treating toxicity of toad venom than digoxin toxicity. This is in agreement with the relatively poor neutralizing ability of digoxin-specific Fab toward cinobufagen and cinobufatolin, which were among constituents of the toad venom in both studies by Brubacher et al. These two bufodienolides are the only inhibitors that required digoxin-specific Fab concentrations several fold higher JPET Fast Forward. Published on February 24, 2004 as DOI: 10.1124/jpet.104.065250 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #65250 19

than their IC50 concentrations to produce neutralization. Other factors in toad venom unrelated to bufodienolides may also be involved in toxicity.

The source of Na/K ATPase used in this study was human cardiac membranes, which demonstrated a more potent inhibition by ouabain than marinobufagenin. This relative potency is similar to that observed for the Downloaded from sarcolemma fraction of the rat thoracic aorta and is the reverse of the relative activity seen in the nerve ending plasmalemma of this tissue (Fedorova and

Bagarov, 1997). Comparison of the ability of several cardenolides to inhibit jpet.aspetjournals.org cardiac Na/K ATPase activity shows that digoxin and digitoxin gain a slight amount of activity over their respective 3 position nonglycosylated forms. This is at ASPET Journals on September 26, 2021 not the case for the cardenolides that are hydroxylated at the 5 position where the rhamnosides of strophanthidin and ouabagenin are 16-fold for convallatoxin and

30-fold for ouabain more potent respectively than their aglycones. The area around the 3 position, where glycosylation occurs, and the adjacent 5 position that is sometimes hydroxylated appear to be important regions for Na/K ATPase inhibitory activity. This is consistent with the recently generated three- dimensional structure-activity relationship study using comparative molecular field analysis alignment (Farr et al., 2002a). Using 28 steroid Na/K ATPase inhibitors, it was found that adding steric bulk and/or electronegativity around the sugar attached to position 3 increased inhibitory activity. The importance of hydroxylation of the 5 position in affecting the spatial orientation of gycosides in the 3 position has recently been show also using molecular field analysis (Farr et JPET Fast Forward. Published on February 24, 2004 as DOI: 10.1124/jpet.104.065250 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #65250 20

al., 2002b). A greater range of inhibitory activity was seen in this study compared to Farr et. al. Perhaps this is because potassium, as opposed to ATP, was added last in these studies which increased ouabain activity greater than 30 fold (data not shown) consistent with current models of the ATPase (Lingrel and

Kuntzweiler, 1994; Yingst et al., 1998).

Downloaded from An unexpected finding in these studies was that the structure activity relationship for the cross-reactivity of digoxin-specific Fab toward the ATPase inhibitors was not always predictive of neutralization activity. ELISA type assays jpet.aspetjournals.org are commonly used to test cross-reactivity and represent a global comparison of how molecules overlap in the antigen binding site of antibodies in a competitive at ASPET Journals on September 26, 2021 manner. While the cross-reactivity of oubain and digoxin was 1.4% based on

ELISA data, digoxin-specific Fab neutralized oubain to the nearly the same extent as digoxin. In addition, 3H-ouabain bound to with nM affinity to digoxin-specific

Fab in the absence of a competitor.

Endogenous Na/K ATPase inhibitors have been demonstrated with physical characteristics similar to ouabain (Hamlyn et al., 1991; Di Bartolo et al.,

1995; El-Masri et al, 2002; Balzan et al., 2000), and bufalin (Hilton et al., 1997).

On the other hand, there is growing evidence that marinobufogenin or a marinobufogenin-like bufodienolide endogenous Na/K ATPase inhibitor exists. A mass spectroscopy study has shown the identity of material isolated from the urine of patients with acute myocardial infarction (Bagrov et al., 1998) to be JPET Fast Forward. Published on February 24, 2004 as DOI: 10.1124/jpet.104.065250 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #65250 21

marinobufogenin. Marinobufogenin specific antibodies were able to identify the material in saline volume expanded anaesthetised dogs (Bagrov et al., 1996).

Marinobufogenin appears to act on a different alpha subunit than ouabain

(Fedorova and Bagrov, 1997) and may stimulate Na/K ATPase activity at low concentrations (Bagarov and Fedorova, 1998). In the present study we found that the neutralizing potency of digoxin-specific Fab towards ouabain and bufalin was Downloaded from similar to that of digoxin and digitoxin and was much greater than marinobufagenin. Because the potency of marinobufagenin in the cardiac membrane ATPase was relatively low compared to ouabain, the effect of digoxin- jpet.aspetjournals.org specific Fab on neutralizing the activity of marinobufagenin at concentrations similar to digoxin and ouabain could not be determined. The heart may be the at ASPET Journals on September 26, 2021 target of an ouabain-like endogenous factor. It appears therefore that human cardiac tissue is more sensitive to ouabain than marinobufagenin and the effect of ouabain can be effectively neutralized by digoxin-specific Fab. JPET Fast Forward. Published on February 24, 2004 as DOI: 10.1124/jpet.104.065250 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #65250 22

Acknowledgements

The authors would like to acknowledge Dr C David Adair, Department of

Obstetrics and Gynecology, Louisianna State University Medical Center, and Dr

Steven W. Graves, Department of Chemistry and Biochemistry, Brigham Young

University, UT for their helpful suggestions and Maria C. McDevitt for secretarial assistance in preparing this manuscript. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 26, 2021 JPET Fast Forward. Published on February 24, 2004 as DOI: 10.1124/jpet.104.065250 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #65250 23

REFERENCES

Adair CD, Buckalew V, Taylor K, Ernest JM, Frye AH, Evans C andVeille JC

(1996) Elevated endoxin-like factor complicating a multifetal second trimester pregnancy: Treatment with digoxin-binding immunoglobulin. Am J Nephrol

16:529-531.

Downloaded from Bagrov AY, Fedorova OV, Dmitrieva RI, French AW and Anderson DE (1996)

Plasma marinobufagenin-like and ouabain-like immunoreactivity during saline volume expansion in anesthetized dogs. Cardiovasc Res 31:296-305. jpet.aspetjournals.org

Bagrov AY,. Fedorova OV, Dmitrieva RI, Howald WN, Hunter AP, Kuznetsova at ASPET Journals on September 26, 2021 EA and Shpen VM (1998) Characterization of a urinary bufodienolide Na+,K+-

ATPase inhibitor in patients after acute myocardial infarction. Hypertension

31:1097-1103.

Bagrov AY and Fedorova OV (1998) Effects of two putative endogenous digitalis-like factors, marinobufagenin and ouabain, on the Na+, K+-pump in human mesenteric arteries. J Hypertens 16:1953-1958.

Balzan S, Montali U, Biver P and Ghione S (1991) Digoxin-binding antibodies reverse the effect of endogenous digitalis-like compounds on Na,K-ATPase in erythrocytes. J Hypertens 9:S304-S305.

JPET Fast Forward. Published on February 24, 2004 as DOI: 10.1124/jpet.104.065250 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #65250 24

Balzan S, D'Urso G, Ghione S, Martinelli A and Montali U (2000) Selective inhibition of human erythrocyte Na+/K+ ATPase by cardiac glycosides and by a mammalian digitalis like factor. Life Sciences 67:1921-1928.

Brubacher JR, Ravikumar PR, Bania T, Heller MB and Hoffman RS (1996)

Treatment of toad venom poisoning with digoxin-specific Fab fragments. Chest Downloaded from 110: 1282-1288.

Brubacher JR, Lachmanen D, Ravikumar PR and Hoffman RS (1999) Efficacy of jpet.aspetjournals.org digoxin specific Fab fragments (Digibind) in the treatment of toad venom poisoning. Toxicon 37:931-942. at ASPET Journals on September 26, 2021

Curd J, Smith TW, Jaton JC and Haber E (1971) The isolation of digoxin-specific antibody and its use in reversing the effects of digoxin. Proc Natl Acad Sci USA

68: 2401-2406.

Dasgupta A and Emerson L (1998) Neutralization of cardiac toxins oleandrin, oleandrigenin, bufalin, and cinobufotalin by digibind: Monitoring the effect by measuring free digitoxin concentrations. Life Sciences 63:781-788.

Di Bartolo V, Balzan S, Pieraccini L,. Ghione S, Pegoraro S, Biver P, Revoltella

R and Montali U (1995) Evidence for an endogenous ouabain-like JPET Fast Forward. Published on February 24, 2004 as DOI: 10.1124/jpet.104.065250 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #65250 25

immunoreactive factor in human newborn plasma coeluted with ouabain on

HPLC. Life Sciences 57:1417-1425.

El-Masri MA, Clark BJ, Qazzaz HM and Valdes R Jr (2002) Human adrenal cells in culture produce both ouabain-like and dihydroouabain-like factors. Clin Chem

48:1720-1730. Downloaded from

Farr CD, Burd C, Tabet MR, Wang X, Welsh WJ and Ball WJ Jr (2002) Three- dimensional quantitative structure-activity relationship study of the inhibition of jpet.aspetjournals.org

Na(+),K(+)-ATPase by cardiotonic steroids using comparative molecular field analysis. Biochemistry 41:1137-1148. at ASPET Journals on September 26, 2021

Farr CD, Burd C, Tabet MR, Ball WJ Jr, Fishwild DM, Wang X, Nair AC and

Welsh WJ (2002) Three-dimensional quantitative structure-activity relationship analysis of ligand binding to human sequence antidigoxin monoclonal anibodies using comparative molecular field analysis. J Med Chem 45 3257-3270.

Fedorova OV and Bagrov AY (1997) Inhibition of Na/K ATPase from rat aorta by two Na/K pump inhibitors, ouabain and marinobufagenin: evidence of interaction with different alpha-subunit isoforms. Am J Hypertension 10:929-935.

JPET Fast Forward. Published on February 24, 2004 as DOI: 10.1124/jpet.104.065250 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #65250 26

Fedorova OV, Kolodkin NI, Agalakova NI, Lakatta EG and Bagrov AY (2001)

Marinobufagenin, an endogenous alpha-1 sodium pump ligand, in hypertensive

Dahl salt-sensitive rats. Hypertension 37:462-466.

Forbush B 3rd (1983) Assay of Na,K-ATPase in plasma membrane preparations:

Increasing the permeability of membrane vesicles using sodium dodecyl sulfate Downloaded from buffered with bovine serum albumin. Anal Biochem 128:159-163.

Fridman AI, Matveev SA, Agalakova NI, Fedorova OV, Lakatta EG and Bagrov jpet.aspetjournals.org

AY (2002) Marinobufagenin, an endogenous ligand of alpha-1 sodium pump, is a marker of congestive heart failure severity. J Hypertension 20:1189-1194. at ASPET Journals on September 26, 2021

Goodlin RC (1988) Antidigoxin antibodies in eclampsia. N England J Med

318:518-519.

Graves SW, Lincoln K, Cook SL and Seely EW (1995) Digitalis-like factor and digoxin-like immunoreactive factor in diabetic women with preeclampsia, transient hypertension of pregnancy, and normotensive pregnancy. Am J

Hypertension. 8:5-11.

Hamlyn JM, Blaustein MP, Bova S, DuCharme DW, Harris DW, Mandel F,

Mathews WR and Ludens JH (1991) Identification and characterization of a JPET Fast Forward. Published on February 24, 2004 as DOI: 10.1124/jpet.104.065250 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #65250 27

ouabain-like compound from human plasma. Proc Natl Acad Sci USA

88:6259-6263.

Hilton PJ, White RW, Lord GA, Garner GV, Gordon DB, Hilton MJ, Forni LG,

McKinnon W, Ismail FM, Keenan M, Jones K and Morden WE (1996) An inhibitor of the sodium pump obtained from human placenta. Lancet 348:303-305. Downloaded from

Huang BS and Leenen FH (1996) Brain "ouabain" and angiotensin II in salt- sensitive hypertension in spontaneously hypertensive rats. Hypertension jpet.aspetjournals.org

28:1005-1012.

at ASPET Journals on September 26, 2021 Kaide J, Ura N, Torii T, Nakagawa M, Takada T and Shimamoto K (1999)

Effects of digoxin-specific antibody Fab fragment (Digibind) on blood pressure and renal water-sodium metabolism in 5/6 reduced renal mass hypertensive rats.

Am J Hypertension 12:611-619.

Kaminski K and Rechberger T (1991) Concentration of digoxin-like immunoreactive substance in patients with preeclampsia and its relation to severity of pregnancy-induced hypertension. Am J Obstet Gynecol 165:733-736.

Ke YS (2001) Endoxin: a major factor regulating cardiovascular system. Acta

Pharmacol Sin 22:201-209.

JPET Fast Forward. Published on February 24, 2004 as DOI: 10.1124/jpet.104.065250 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #65250 28

Krep HH, Graves SW, Price DA, Lazarus M, Ensign A, Soszynski PA and

Hollenberg NK (1996) Reversal of sodium pump inhibitor induced vascular smooth muscle contraction with digibind. Stoichiometry and its implications. Am

J Hypertension 9:39-46.

Levi AJ, Boyett MR and Lee CO (1995) The cellular actions of digitalis Downloaded from glycosides in the heart. Prog Biophys Mol Biol 62:1-54.

Li M, Wen C and Whitworth JA (1997) Hemodynamic effects of the Fab jpet.aspetjournals.org fragment of digoxin antibody (digibind) in corticotropin (ACTH)-induced hypertension. Am J Hypertension 10:332-336. at ASPET Journals on September 26, 2021

Lingrel JB and Kuntzweiler T (1994) Na+,K(+)-ATPase. J Biol Chem

269:19659-19662.

Lopatin DA, Ailamazian EK, Dmitrieva RI, Shpen VM, Fedorova OV, Doris PA and Bagrov AY (1999) Circulating bufodienolide and cardenolide sodium pump inhibitors in preeclampsia. J Hypertension 17:1179-1187.

Melero CP, Medarde M and San Fliciano A (2000) A short review on cardiotonic steroids and their aminoguanadine analogues. Molecules 5:51-81.

JPET Fast Forward. Published on February 24, 2004 as DOI: 10.1124/jpet.104.065250 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #65250 29

Morris RG, Frewin DB, Saccoia NC, Goldsworthy WL, Jeffries WS and McPhee

AJ (1990) Interference from digoxin-like immunoreactive substance(s) in commercial digoxin kit assay methods. Eur J Clin Pharmacol 39:359-63.

Naomi S, Graves S, Lazarus M, Williams GH and Hollenberg NK (1991)

Variation in apparent serum digitalis-like factor levels with different digoxin Downloaded from antibodies. The "immunochemical fingerprint". Am J Hypertension 4:795-801.

Oda M, Kurosawa M, Numazawa S, Tanaka S, Akizawa T, Ito K, Maeda M and jpet.aspetjournals.org

Yoshida T (2001) Determination of bufalin-like immunoreactivity in serum of humans and rats by time-resolved fluoroimmunoassay for using a monoclonal at ASPET Journals on September 26, 2021 antibody. Life Sciences 68:1107-1117.

Shild HO (1947) pAx and competitive drug antagonism. Br JPharmacol

4:277-280.

Sich B, Kirch U, Tepel M, Ziedek W, and Schoner W (1996) Pulse pressure correlates with a proscillardin A immunoreactive compound. Hypertension

27:1073-1078.

Schoner W (2002) Endogenous cardiac glycosides, a new class of steroid hormones. Eur J Biochem 269:2440-2448.

JPET Fast Forward. Published on February 24, 2004 as DOI: 10.1124/jpet.104.065250 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #65250 30

Tao QF, Soszynski PA, Hollenberg NK and Graves SW (1996) Specificity of the volume-sensitive sodium pump inhibitor isolated from human peritoneal dialysate in chronic renal failure. Kidney Int 49:420-429.

Teruya H, Yamazato M, Muratani H, Sakima A, Takishita S, Terano Y and

Fukiyama K (1997) Role of ouabain-like compound in the rostral ventrolateral Downloaded from medulla in rats. J Clin Investig 99:2791-2798.

Yingst DR, Yang SY and Schiebinger R (1998) Purification of active Na+-K+- jpet.aspetjournals.org

ATPase using a new ouabain-affinity column. Am J Physiol 275:C1167-C1177. at ASPET Journals on September 26, 2021 JPET Fast Forward. Published on February 24, 2004 as DOI: 10.1124/jpet.104.065250 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #65250 31

FIGURE LEGENDS

Figure 1. Steroid structure of cardeniolide Na/K ATPase inhibitors.

Cardenolides have a five-membered lactone ring while the

bufodienolides have a six-membered lactone ring.

Figure 2. Concentration related inhibition of Na/K ATPase by cardeniolide Downloaded from inhibitors. Panel A compares activity of glycosides and aglycones

of steroids lacking a hydroxyl in the 5 position. Panel B compares

5-hydroxyl cardenolides with (closed symbols) and without (open jpet.aspetjournals.org

symbols) a rhamnose in the 3 position. (n=3)

at ASPET Journals on September 26, 2021 Figure 3. Eleven concentrations digoxin-specific Fab ranging from 0.1 uM

to 2.5 uM shift the Na/K ATPase inhibition curve of

marinobufogenin toward the right (panel A) A Schild plot is

generated form the IC50 values (Panel B).

Figure 4. Cardenolides and bufodienolides compete for binding of 3H-

ouabain to digoxin-specific Fab coated assay plates. (n=3)

Figure 5. Ability of several Na/K ATPase inhibitors to compete with

digoxin-specific Fab binding to digoxin-BSA coated wells in an

ELISA type assay. (n=3) JPET Fast Forward. Published on February 24, 2004 as DOI: 10.1124/jpet.104.065250 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #65250 32

Table 1. Substitutions on various positions of the general steroid structure of cardenolide and bufodienolide Na/K ATPase inhibitors.

1 3 5 10 11 12 14 16

Cardenolides

Digoxigenin OH CH3 OH OH

Digoxin (Dig)3 CH3 OH OH

Digitoxigenin OH CH3 OH Downloaded from

Digitoxin (Dig)3 CH3 OH

Neriifolin Thevose CH3 OH jpet.aspetjournals.org Strophanthidin OH OH OH

Convallatoxin Rhamnose OH OH

Acetylstrophanthidin Acetyl OH OH

Ouabagenin OH OH OH OH OH at ASPET Journals on September 26, 2021

Ouabain OH Rhamnose OH OH OH

Bufodienolides

Bufalin OH OH

Proscillardin Rhamnose OH

Marinobufagenin OH OH 14-O-15

Cinobufagen OH 14-O-15 Acetyl

Cinobufatolin OH OH 14-O-15 Acetyl

JPET Fast Forward. Published on February 24, 2004 as DOI: 10.1124/jpet.104.065250 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #65250 33

Table 2. Summary of Digoxin-specific Fab characterization data, mean and (+/-standard error), for four types of experiments

ATPase IC50 Digoxin – 3H-Ouabain ELISA IC50

nM specific Fab nM nM

Neut.

nM Downloaded from

Digoxigenin 160 (+/-9) 200 (+/-52) 0.19 (+/-0.07) 14 (+/-1)

Digoxin 120 (+/-20) 15 (+/-1) 0.51 (+/-0.16) 41 (+/-10)

Digitoxigenin 130 (+/-53) 79 (+/-8) 0.60 (+/-0.21) 96 (+/-21) jpet.aspetjournals.org

Digitoxin 78 (+/-18) 8.4 (+/-2.1) 2.5 +/-0.6) 54 (+/-14)

Neriifolin 12 (+/-1) 12 (+/-1) 0.66 (+/-0.16) 55 (+/-12)

Strophanthidin 180 (+/-7) 190 (+/-5) 6.7 (+/-2.0) 7100 (+/-1400) at ASPET Journals on September 26, 2021

Convallatoxin 11 (+/-1) 6.9 (+/-1) 0.36 (+/-0.1) 720 (+/-290)

Acetylstrophanthidin 140 (+/-20) 60 (+/-9) 0.64 (+/-0.16) 2100 (+/-300)

Ouabagenin 640 (+/-90) 580 (+/-52) 14 (+/-3) 16000 (+/-2000)

Ouabain 21 (+/-1) 20 (+/-2) 3.9 (+/-0.8) 2900 (+/-700)

Bufalin 53 (+/-6) 13 (+/-1) 1.8 (+/-0.4) 690 (+/-87)

Proscillardin 130 (+/-15) 30 (+/-2) 5.6 (+/-1.0) 2900 (+/-410)

Marinobufagenin 520 (+/-49) 470 (+/-30) 53 (+/-12) 6900 (+/-2100)

Cinobufagen 50 (+/-4) 420 (+/-14) 200 (+/-6) 7400 (+/-2300)

Cinobufatolin 160 (+/-4) 1400 (+/-48) 4800 (+/-600) >100000

This articlehasnotbeencopyeditedandformatted.Thefinalversionmaydifferfromthisversion.

O JPET FastForward.PublishedonFebruary24,2004asDOI:10.1124/jpet.104.065250

O

CH3

12 17 11 13 16 14 1 9 15 2 10 8

3 5 7

4 6

Downloaded from from Downloaded jpet.aspetjournals.org at ASPET Journals on September 26, 2021 26, September on Journals ASPET at JPET Fast Forward. Published on February 24, 2004 as DOI: 10.1124/jpet.104.065250 This article has not been copyedited and formatted. The final version may differ from this version.

A 100 Digoxigenin 80 Digoxin Digitoxigenin 60 Digitoxin Activity 40 20 % Control ATPase

0 Downloaded from -9 -8 -7 -6 -5 Conc. Log(M) jpet.aspetjournals.org

B 100 Strophanthidin at ASPET Journals on September 26, 2021 80 Convallatoxin Ouabagenin 60 Ouabain

Activity 40 20 % Control ATPase 0 -10 -9 -8 -7 -6 -5 Conc. Log(M)

Figure 2 JPET Fast Forward. Published on February 24, 2004 as DOI: 10.1124/jpet.104.065250 This article has not been copyedited and formatted. The final version may differ from this version.

A 100 80 60

Activity 40

20 Downloaded from % Control ATPase Control % 0 -8 -7 -6 -5 -4

Marinobufagenin Log(M) jpet.aspetjournals.org

B

0.6 at ASPET Journals on September 26, 2021

0.3

0.0

-0.3 Log(Dose Ratio-1) Log(Dose -0.6 5 6 7 8 -Log[Digoxin-specific Fab]

Figure3 JPET Fast Forward. Published on February 24, 2004 as DOI: 10.1124/jpet.104.065250 This article has not been copyedited and formatted. The final version may differ from this version.

Ouabain 100 Digoxin 80 Strophanthidin 60 Convallatoxin

Bound Marinobufogenin 40 Cinobufatolin % 3H-Ouabain 20 Downloaded from 0 -11 -10 -9 -8 -7 -6 -5 -4

Conc. Log(M) jpet.aspetjournals.org at ASPET Journals on September 26, 2021

Figure4 JPET Fast Forward. Published on February 24, 2004 as DOI: 10.1124/jpet.104.065250 This article has not been copyedited and formatted. The final version may differ from this version.

Digoxin 100 Digitoxin 80 Convallatoxin Strophanthidin 60 Bufalin 40 Downloaded from % Bound % Marinobufagenin 20 0 -10 -9 -8 -7 -6 -5 -4 -3 jpet.aspetjournals.org Conc. Log(M) at ASPET Journals on September 26, 2021

Figure 5