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Biotransformation and pharmacokinetic overview of enoximone and its sulfoxide metabolite
Biotransformation and Pharmacokinetic Overview
RICHARD
A. OKERHOLM, PhD, KENNETH Y. CHAN, MA, JAMES F. LANG, GARY A. THOMPSON, PhD, and STEPHEN J. RUBERG, PhD
Enoximone possesses both positive inotropic and vasodilatory activities and may be useful in the treatment of patients with congestive heart failure (CHF). In all animal species investigated (rat, dog, monkey and man), the major urinary metabolite is the sulfide oxidation product (sulfoxide); very little unchanged drug appears in urine. Both in vitro and in vivo animal studies indicate reversibility of the sulfoxidation reaction; therefore, it is presumed that sulfoxidation is reversible in man. In normal healthy subjects, no difference in extent of absorption due to dietary state is observed. In patients with New York Heart Association class Ill to IV CHF, median
E
noximone is a member of a new class of cardiotonic agents. It is neither a cardiac glycoside nor sympathomimetic in nature and is intended for intravenous and oral administration. AnimallJ and human3-5 studies have demonstrated that enoximone possesses both positive inotropic and vasodilatory activities; both actions should be beneficial in the treatment of patients with congestive heart failure (CHF]. Enoximone is primarily eliminated via sulfoxidation. Although less potent, the sulfoxide metabolite also possesses positive inotropic and vasodilatory properties. This article summarizes the biotransformation and pharmacokinetic studies conducted on enoximone.
Drug Metabolism The biotransformation pathways for enoximone are qualitatively similar among investigated species. Figure 1 illustrates chemical structures and mean urinary recoveries of intact drug and metabolites after intravenous administration to male rats, dogs, cynomolgus monkeys and humans. Intact drug and the sulfone metabolite are only present in small amounts. From the Drug Metabolism Department, Merrell Dow Research Institute, Cincinnati, Ohio, Address for reprints: Richard A. Okerholm, PhD, Drug Metabolism Department, Merrell Dow Research Institute, 2110 East Galbrairh Road, Cincinnati, Ohio 45215-6300.
MS,
terminal disposition half-lives for enoximone and its sulfoxide metabolite are 6.2 to 7.6 hours, respectively; Enoximone and sulfoxide plasma concentrations from high dose intravenous infusion studies in patients with class Ill to IV CHF were also investigated. The collective data suggest nonlinearity in one or more pharmacokinetic processes, of which one may be saturation of sulfoxidation. No direct relation between enoximone and/or the sulfoxide metabolite plasma concentration and pharmacologic effect has been established. (Am J Cardiol
1987;60:21C-28C)
The dog is the only species found to excrete a major fraction of the dose as &methylthiohippuric acid. In all species studied, the sulfoxide metabolite is the major moiety present in plasma and is also the predominant urinary excretion product. In healthy men, 8 hour urinary sulfoxide recovery averages 78% of an oral solution dose. Although synthetic enoximone sulfoxide exists as 2 stereoisomers (asymmetric sulfur), the sulfoxidation reaction in rats, monkeys and humans is stereospecific. To date, absolute configuration of the sulfoxide isomer formed (which is the same for all 3 species] has not been determined. Unlike the aforementioned species, however, the dog excretes both stereoisomers in approximately equal proportions. Sulfoxide formation and elimination is a very important biotransformation pathway in man. It is found at high levels in plasma after either intravenous or oral enoximone administration, and in animal tests (and presumably therefore in humans), it possesses cardiotonic activity. Using a synthetic racemic mixture, enoximone sulfoxide produced concentration-dependent inotropic effects in the guinea pig and cat left atria1 strip preparations as well as the cat papillary muscle preparation. Sulfoxide to enoximone potency ratios were approximately 0.41,0.26 and 0.079, respectively. In pentobarbital-barbital anesthetized dogs, intravenous injection of racemic enoximone sulfoxide produced a qualitatively similar inotropic effect one-
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seventh as potent as enoximone but with a 13 times longer duration of action.6 After intravenous administration of enoximone to dogs, the sulfoxide area under the plasma concentration-time curve averaged 1.68 times greater than the intact drug area under the plasma concentration-time curve. After intravenous administration of racemic enoximone sulfoxide to dogs, the sulfoxide:enoximone area under the plasma concentration-time curve ratio averaged 18.1. Absolute in vivo determination of potency is difficult, because the oxidation of enoximone to sulfoxide as well as the reduction of sulfoxide to enoximone are reversible reactions. Reversibility of metabolism was also observed in rats. Figure 2 shows enoximone and sulfoxide plasma and whole heart concentration-time profiles after intravenous enoximone administration to rats. Figure 3 shows these profiles after intravenous administration of racemic enoximone sulfoxide. In vitro studies have also been conducted. The oxidation of enoximone to sulfoxide was investigated in rat liver and kidney 9,000 g supernatant fractions (Fig. 4). The reduction of sulfoxide was studied in rat liver and kidney 9,006 g supernatant fractions [Fig. 4). It is clear that oxidative enoximone metabolism and reductive sulfoxide metabolism each occurs in both liver and kidney preparations. In these in vitro studies, the liver was more active than the kidney in oxidation, whereas reduction was more active in the kidney. One may speculate that reversible metabolism also occurs in humans.
directly from enoximone in the kidney, the ratio of sulfoxide rate of excretion to its plasma level would not constitute a true renal clearance.7 This may be the case in humans; it was not uncommon to observe intrasubject sulfoxide (rate of excretion/plasma concentration] ratios fluctuating lo- to 15-fold over the course of a study. Additionally, when reversible metabolism is present, intact drug clearance estimates based solely on dose/area under the plasma concentration-time curve ratios of intact drug will be underestimated.*!9 Since we assume that the reversibility of metabolism observed in rats and dogs also occurs in humans, clearance and volume,of distribution cannot unambiguously be determined unless the presumed reversibly formed metabolite is also administered. Because this could not be accomplished under the present Investigational New Drug Application, these reported parameters are only apparent. Another complicating factor in the pharmacokinetic analysis is the apparent nonlinearity. Protein binding: Preliminary studies on enoximone protein binding using serum from healthy adult men indicate that binding is approximately 85% at therapeutic drug concentrations. Single dose oral study in healthy human subjects: To determine the effect of food on the relative bioavailability of enoximone from a soft gelatin capsule formulation (150 mg], a X-way nonrandomized crossover study was conducted in 12 healthy volunteers. In the first period of the study, fasting subjects received a single enoximone capsule with 200 ml of water at 8 A.M. In the second period of the study [l week later), the same subjects consumed a standard breakfast after an overnight fast; the meal, which was served at 7 A.M.,
Pharmacokinetics If in humans (or any other species), significant amounts of sulfoxide excreted in urine are formed
3 IL--
W+;’
H
No
Rat - trace Dog - 1.3% Monkey - 0.8% Man - trace
NH
ENOXIMONE
/
\
CH5S--@C-0~ j
‘d
i
+ [H3cfxo
j
C&
%$;;i;pa3,
SULFOXIDE
(MDL
Man
19,438)
- 78%
GLYCINE I f,--NHCH+OOH 0 GLYCINE CONJUGATE (4-methyithiohippuric
acid)
Rot - detected Dog - 30% Monkey - 5-10X Man - trace
I
V
H N
C LTLrf
C+-
‘d SULFONE
(MDL
o NH
Rat - 5.5% Dog - trace Monkey - trace Man - trace
19,437)
FIGURE 1. Known metabolic pathways for enoximone (bracketed compounds are postulated intermediates). average urinary recoveries. Rat data were obtained from 24-hour urine collections after intravenous 5 mg/kg doses Sprague Dawley (CD) rats. Dog data were obtained from 24-hour urine collections after intravenous 10 mg/kg doses Monkey data were obtained from 24-hour urine collections after intravenous 15 mg/kg doses to 3 male cynomolgus were obtained from 8-hour urine collections after 2 mg/kg oral solution doses to 8 healthy male subjects who Chemical entities were identified and quantitated using sensitive and specific high performance liquid chromatography raphy-mass spectrometry.
Percentage values are to 5 male Charles River to 3 male beagle dogs. monkeys. Human data had fasted overnight. and gas chromatog-
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FIGURE 2. Plasma and whole heart enoximone and sulfoxide concentration-time profiles after intravenous bolus injections of 5 mg/ kg enoximone to male Charles River Sprague Dawley (CD) rats. Two rats each were killed at 5, 15, 30 and 60 minutes: average concentration values were used. A single rat was killed each at 120 and 160 minutes (heart sulfoxide concentrations were undetectable at 160 minutes). Drug and metabolite levels were measured by an accurate and specific high performance liquid chromatographic method.
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FIGURE 3. Plasma and whole heart enoximone and sulfoxide concentration-time profiles after intravenous bolus injections of 5 mg/ kg racemic enoximone sulfoxide to male Charles River Sprague Dawley (CD) rats. Two rats each were killed at 5, 15, 30 and 60 minutes; average concentration values were used. A single rat was killed each at 120 and 160 minutes (concentrations of enoximone and sulfoxlde were undetectable at 160 mlnules). Drug and metabolite levels were measured by an accurate and specific high performance liquid chromatographic method.
Liver 2s
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Kidnev
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consisted of cereal, skim milk, a banana, toast, jelly, margarine (1 pat) and decaffeinated coffee with sugar. At 8 A.M., the subjects received 150 mg enoximone (capsule) with 200 ml of water. During both periods of the study, lunch was provided at 12 noon. Blood [disodium ethylenediaminetetraacetic acid anticoagulant) was obtained at 0, 0.5, 1, 1.5, 2, 3, 4, 6, 8, 10, 12, 16 and 24 hours. Plasma was separated and frozen until analysis. Urine samples were collected at -12 to 0,O to 22 to 4,4 to 6, 6 to 8, 8 to 10, 10 to 12, 12 to 16 and 16 to 24 hours. Urine volumes were measured, and aliquots were frozen until analysis. Plasma enoximone and sulfoxide concentrations were measured by a specific, sensitive and accurate high performance liquid chromatography procedure. lo A modified procedure was used to determine urine concentrations of sulfoxide. Figure 5 shows mean enoximone and enoximone sulfoxide plasma concentration-time profiles and Table I summarizes mean pharmacokinetic parameter estimates. Statistically significant differences between the nonfasting and fasting conditions were observed (p
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90
,
FIGURE 4. Enoxlmone sulfoxlde (/err) and enoximone (right) formation after incubation of 5 X 10e6 M (1.24 mg/liter) enoximone with male Charles River rat liver and kidney 9,000 g superna-
120
(MINUTES)
<0.05) for enoximone and sulfoxide peak concentrations but not for time of peak concentration, area under the plasma concentration-time curve (0 to 24 hours] and urinary sulfoxide recovery. Although the power to detect a 20% difference [a = 0.05) due to dietary state for time of peak concentration was low, it was greater than 0.90 for all other parameters that showed no difference. These results indicate that although the presence of food affects the rate of drug absorption (via altering drug absorption and/or hepatic blood flow], overall the extent of bioavailability is not appreciably influenced. Multiple dose oral study in healthy human subjects: In a limited 2-way crossover study in 8 normal male volunteers, the relative bioavailability of enoximone from the soft gelatin capsule formulation was compared with that of a solution formulation (5 mg/ ml]. During the first period of the study, subjects received 150 mg enoximone orally every 8 hours [around the clock] for 22 doses; subjects were randomly allocated to formulations. After a z week washout, the sub-
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UYt
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tNOXlMONE
TABLE I Pharmacokinetic of Enoxlmone Absorption
IN HEART
FAILURE
Parameter Estimates Under Fasting Conditions
Obtained from a 12 Subject Bioavailability Study and After a Standardized Breakfast (nonfasting) Enoximone
Enoximone Parameter AUC [(rig/ml) . hr] (0 to 24 hours) Peak concentration @g/ml) Time to peak (hr) Cumulative percent urinary recovery (0 to 24 hours)
Fasting 551
f
Nonfasting 169
569
f
Sulfoxide
Fasting 119
81 f36
123 f 2%
2.5 f 2.6 not assayed
1.3 f 0.40 not assayed
4,311 663
Nonfasting
f 491
4,605
f
i 246
1,130
f 230
1.5 f 0.64 75 f 13*
* Data available for 10 subjects because of incomplete urine collections. Data are mean f standard deviation; enoximone was administered as 150 mg in a soft gelatin AUC = area under the plasma concentration-time curve. Mean plasma concentration-time curves are shown in Figure 5.
1,095
1.6 f 0.66 76 f 0.7
capsule.
0 T,ME4vi0& FIGURE 5. Mean enoximone (fop) and enoximone sulfoxide (boftom) plasma concentration-time profiles after oral administration of 150 mg enoximone in a soft gelatin capsule formulation (fasting and nonfasting conditions). The nonfastlng condition denotes that the capsule was ingested after consumption of a standardized breakfast (see text). Mean parameter estimates are summarized in Table I.
jects were crossed over, and the protocol was repeated. The first dose of each day was always administered immediately after breakfast. Solution doses (30 ml of the 5 mg/ml solution) were administered in 4 to 6 ounces of fruit juice; capsules were ingested with 200 ml of water. BIood samples (disodium ethylene diaminetetraacetic acid anticoagulant) were obtained at: (1) 0,0.25,0.5,1,2,3,4,6 and 8 hours after the first dose; (2) immediately before the morning dose on days 2
1’2 1’6 2’0 2’4
FIGURE 6. Mean enoximone and enoximone sulfoxide plasma concentration-time profiles after single and multiple oral enoximone doses (150 mg) to healthy male subjects; two dosage forms were studied, a solution and a soft gelatin capsule. Drug was administered at 8 hour intervals (around the clock) (see text).
through 7; (3) 0, 0.25,0.5, 1, 2,3,4, 6,8, 10, 12, 18 and 24 hours after the last dose (22) on day 8. Urine samples were collected over 8 hour intervals after the first 3 doses on day 1. On days 2 through 7, M-hour urine collections were obtained. On the morning of day 8 [with dose 22), urine was collected for 3 sequential 8 hour periods. Blood was centrifuged and harvested for its plasma; both plasma and urine specimens were treated as previously described. Whereas only sulfoxide was analyzed in urine, both enoximone and sulfox-
August
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ide were measured in plasma (the same analytical methods were used as in the single dose, food effect study]. Figure 6 illustrates mean enoximone and enoximone sulfoxide plasma concentration-time profiles. Considerable scatter in the data after the last dose made it impossible to estimate accurately the terminal exponential half-life. Statistical analysis of the data indicated that although there were differences in rate of enoximone absorption between the z formulations, the relative extent of bioavailability did not differ by more than 5% to 1070. What is perhaps most clinically relevant is the accumulation of enoximone and its sulfoxide metabolite. Wagner l1 defined the drug concentration index (R,) of accumulation as:
R, =
average plasma concentration 7 hours at steady-state
during
average plasma concentration from 0 to 7 hours after the first dose where 7 is the dosing interval. Using the data after administration of the solution, the mean drug concentration index was 2.1 for enoximone and 1.5 for sulfoxide. The mean steady-state sulfoxide:enoximone plasma concentration ratio (determined from dose 22 and adjusted for molecular weight) was 3.9. This latter value was considerably less than the mean ratio of sulfoxide:enoximone single-dose areas under the plasma concentration-time curves (0 to 24 hours) observed in fasting subjects in the previously described bioavailability study. The mean value obtained in that study (corrected for molecular weight] was 7.7. Viewed collectively, these data suggest nonlinearity of 1 or more pharmacokinetic processes, which may be saturability of sulfoxidation. Intravenous singie-dose studies in class III and IV congestive heart failure patients: Enoximone was administered by intravenous infusion (2.8 to 16.6 min-
utes] to 6 patients (5 male, 1 female) with severe (class III or IV] CHF. Five patients received 2 different doses, ranging from 0.5 to 3.0 mg/kg. Figure 7 shows enoximone and sulfoxide plasma concentration-time profiles for a representative subject. The terminal exponential half-life for enoximone ranged from 3.0 to 8.1 hours (median 6.2); for the sulfoxide metabolite, the half-life ranged from 6.0 to 11.0 hours (median 7.6). Area under the metabolite curve ranged from 2.9 to 8.3 (median 4.8) times greater than for intact drug (corrected for molecular weight]. Apparent total clearance for enoximone ranged from 3.7 to 13.0 (median 9.61 ml/ min/kg. Apparent steady-state volume of distribution ranged from 2.1 to 8.0 (median 4.2) liters/kg. Over a 24hour period, a median 0.49% of the dose was recovered in urine as intact drug. The median recovery of sulfoxide in a 24 hour urine was 74%. Both intra- and intersubject apparent renal clearance for sulfoxide varied considerably, suggesting enoximone and sulfoxide may have been interconverted by the kidney. In the 5 subjects receiving 2 different doses, the area ratio of sulfoxide to intact drug was greater than the lower dose. The median percent increase was 42%. Although the sample size does not permit extensive sta-
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tistical analysis, these data are consistent with dosedependent kinetics in the sulfoxidation reaction, It was not possible to correlate drug/metabolite plasma concentration to pharmacodynamic response, possibly due in part to the fact that the subjects were receiving concomitant medication (particularly digoxin and diuretics). However, some pharmacodynamic responses (systemic vascular resistance, pulmonary vascular resistance and pulmonary capillary wedge pressure) generally decreased after drug administration, whereas cardiac output generally increased. The magnitude of drug responses, however, differed considerably among subjects. Intravenous infusion study in class III to IV congestiye heart failure patients: To investigate the phar-
macokinetics of enoximone and its sulfoxide metabolite during intravenous infusion, plasma concentrations of enoximone and sulfoxide were determined in 12 male patients with severe CHR (class III to IV] who received enoximone therapy. The endximone infusion rate was initially 90 pg/min/kg body weight; once a desired response was achieved (0.42 to 1.0 hour), the infusion rate was reduced to a maintenance rate. The maintenance infusion rates ranged from 7.4 to 40 pg/min/kg, with a median value of 14 pg/min/kg
3000 2000 F = E G c
IOOOBOO-
z 0
soo-
5
300-
a: +
200-
5 s
100-
$j
so-
r”
so-
* 4 n
3020-
lOLl
’ 0
0 2
1
’ 4
HOURS
’
1 6
1
1
1
1 10
:
I
12
(post-infusioi)
FIGURE 7. Enoximone and enoximone sulfoxide plasma concentration-time profiles in a representative patient with class IV congestive heart faiiure who received a 1.5 mg/kg enoximone dose infused over a period of 8.58 minutes (this patient was also receiving heparin, insulin, digoxin, oxycodone and acetaminophen). The so/id and dashed lines are theoretical curves generated from the 4exponential enoximone and sulfoxide plasma concentration-time functions, which were fitted to the postinfusion data; each value of the dependent variable was weighted by the reciprocal of the observed plasma concentration squared.
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data suggests that nonlinear pharmacokinetic processes may have been operative. In summary, the data presented demonstrate that enoximone is readily absorbed and undergoes firstpass metabolism to an active sulfoxide whose formation is saturable. Further, its metabolites are cleared by urinary excretion.
References
I
9
.
l 0
4
8
12
16
20
24
26
32
36
40
44
48
52
56
60
7 64
TIME(HOURS)
FIGURE 8. Plasma concentration-time profiles of enoximone and enoximone sulfoxide in a representative patient with class IV congestive heart failure who received a loading enoximone infusion of 5.1 mg/min from 0 to 0.68 hours and a maintenance infusion of 2.3 mg/min from 0.77 to 45 hours.
(there was a 0.083 hour lag between the loading and maintenance infusion). The maintenance infusion lasted 29 to 48 hours. Figure 8 illustrates representative enoximone and enoximone sulfoxide plasma concentration-time profiles in 1 patient. It should be noted that although the plasma concentrations of enoximone and its sulfoxide metabolite did not reach steady-state in all patients [the reason for this is currently unknown, but may have been associated with the severity of disease], others did appear to reach steady-state within the first 24 hours of infusion. Nonetheless, the fact that steady-state was not achieved in all of the patients within the time predicted from single dose
1. Dage RC, Roebel LE, Hsieh CP, Weiner DL, Woodward JK. Cardiovascular properties of a new cardiotonic agent: MDL 17,043 (1,3-dihydro-4-methy&[4-(methylthio)-benzoyIJ-2H-imidazol-2-one]. J Cardiovasc Pharmacol 1982; 4:500-508. 2. Roebel LE, Dage RC, Cheng HC, Woodward JK. Characterization of the cardiovascular activities of a new cardiotonic agent MDL 17,043 (1,3-dihydro4-methyI-6-[4-(methyIthio)-benzoy~-2H-imidazoI-2-one), J Cardiovasc Pharmacol 1982;4:721-729. 3. Arbogast R, Brandt C, Haegle KD, Fincker JL, Schechter PJ. Hemodynamic effects of MDL 17,043, a new cardiotonic agent, in patients with congestive heart failure: comparison with sodium nitroprusside. J Cardiovasc Pharmacol 1983;5:998-1004. 4. Crawford MH, Richards KL, Sodums MT, Kennedy GT. Positive inotropic and vasodilator effects of MDL 17,043 in patients with reduced left ventricular performance. Am J Cardiol 1984;53:1051-1053. 5. Amin DK, Shah PK, H&e S, Shellock F. Comparative acute hemodynamic effects of intravenous sodium nitroprusside and MDL 17,043, a new inotropic drug with vasodilator effects, in refractory congestive heart failure. Am Heart J 1985;109:1006-1012. 6. Dage RC, Kariya T, Hsieh CP, Roebel LA, Cheng HS, Schnettler RA, Grisar JM. Pharmacology of enoximone. Am J Cardiol 1987;68:18C-14C. 7. Wan SH, Riegelman S. Renal contribution to overall metabolism of drugs. I: conversion of benzoic acid to hippuric acid. J Pharm Sci 1972;61:1278-1284. 8. Hwang S, Kwan KC, Albert KS. A linear model of reversible metabolism and its application to bioavailability assessment. [ Pharmacokinet Biopharm 1981;9:693-709. 9. Wagner JG, DiSanto AR, Gillespie WR, Albert KS. Reversible metabolism and pharmacokinetics: application to prednisone-prednisolone. Res Comm Chem Pathol Pharmacol 1981;32:387-406. 10. Chan KY, Ohlweiler DF, Lang JF, Okerholm RA. Simultaneous analysis of a new cardiotonic agent, MDL 17,043, and its major sulfoxide metabolite in plasma by high-performance liquid chromatography. J Chromatogr 1984;306: 249-256. 11. Wagner JG. Drug accumulation. 1 Clin Pharmacol 1967;7:84-88.
RICHARD
A. OKERHOLM, PhD, KENNETH Y. CHAN, MA, JAMES F. LANG, GARY A. THOMPSON, PhD, and STEPHEN J. RUBERG, PhD
Enoximone possesses both positive inotropic and vasodilatory activities and may be useful in the treatment of patients with congestive heart failure (CHF). In all animal species investigated (rat, dog, monkey and man), the major urinary metabolite is the sulfide oxidation product (sulfoxide); very little unchanged drug appears in urine. Both in vitro and in vivo animal studies indicate reversibility of the sulfoxidation reaction; therefore, it is presumed that sulfoxidation is reversible in man. In normal healthy subjects, no difference in extent of absorption due to dietary state is observed. In patients with New York Heart Association class Ill to IV CHF, median
E
noximone is a member of a new class of cardiotonic agents. It is neither a cardiac glycoside nor sympathomimetic in nature and is intended for intravenous and oral administration. AnimallJ and human3-5 studies have demonstrated that enoximone possesses both positive inotropic and vasodilatory activities; both actions should be beneficial in the treatment of patients with congestive heart failure (CHF]. Enoximone is primarily eliminated via sulfoxidation. Although less potent, the sulfoxide metabolite also possesses positive inotropic and vasodilatory properties. This article summarizes the biotransformation and pharmacokinetic studies conducted on enoximone.
Drug Metabolism The biotransformation pathways for enoximone are qualitatively similar among investigated species. Figure 1 illustrates chemical structures and mean urinary recoveries of intact drug and metabolites after intravenous administration to male rats, dogs, cynomolgus monkeys and humans. Intact drug and the sulfone metabolite are only present in small amounts. From the Drug Metabolism Department, Merrell Dow Research Institute, Cincinnati, Ohio, Address for reprints: Richard A. Okerholm, PhD, Drug Metabolism Department, Merrell Dow Research Institute, 2110 East Galbrairh Road, Cincinnati, Ohio 45215-6300.
MS,
terminal disposition half-lives for enoximone and its sulfoxide metabolite are 6.2 to 7.6 hours, respectively; Enoximone and sulfoxide plasma concentrations from high dose intravenous infusion studies in patients with class Ill to IV CHF were also investigated. The collective data suggest nonlinearity in one or more pharmacokinetic processes, of which one may be saturation of sulfoxidation. No direct relation between enoximone and/or the sulfoxide metabolite plasma concentration and pharmacologic effect has been established. (Am J Cardiol
1987;60:21C-28C)
The dog is the only species found to excrete a major fraction of the dose as &methylthiohippuric acid. In all species studied, the sulfoxide metabolite is the major moiety present in plasma and is also the predominant urinary excretion product. In healthy men, 8 hour urinary sulfoxide recovery averages 78% of an oral solution dose. Although synthetic enoximone sulfoxide exists as 2 stereoisomers (asymmetric sulfur), the sulfoxidation reaction in rats, monkeys and humans is stereospecific. To date, absolute configuration of the sulfoxide isomer formed (which is the same for all 3 species] has not been determined. Unlike the aforementioned species, however, the dog excretes both stereoisomers in approximately equal proportions. Sulfoxide formation and elimination is a very important biotransformation pathway in man. It is found at high levels in plasma after either intravenous or oral enoximone administration, and in animal tests (and presumably therefore in humans), it possesses cardiotonic activity. Using a synthetic racemic mixture, enoximone sulfoxide produced concentration-dependent inotropic effects in the guinea pig and cat left atria1 strip preparations as well as the cat papillary muscle preparation. Sulfoxide to enoximone potency ratios were approximately 0.41,0.26 and 0.079, respectively. In pentobarbital-barbital anesthetized dogs, intravenous injection of racemic enoximone sulfoxide produced a qualitatively similar inotropic effect one-
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A SYMPOSIUM:
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IN HEART
FAILURE
seventh as potent as enoximone but with a 13 times longer duration of action.6 After intravenous administration of enoximone to dogs, the sulfoxide area under the plasma concentration-time curve averaged 1.68 times greater than the intact drug area under the plasma concentration-time curve. After intravenous administration of racemic enoximone sulfoxide to dogs, the sulfoxide:enoximone area under the plasma concentration-time curve ratio averaged 18.1. Absolute in vivo determination of potency is difficult, because the oxidation of enoximone to sulfoxide as well as the reduction of sulfoxide to enoximone are reversible reactions. Reversibility of metabolism was also observed in rats. Figure 2 shows enoximone and sulfoxide plasma and whole heart concentration-time profiles after intravenous enoximone administration to rats. Figure 3 shows these profiles after intravenous administration of racemic enoximone sulfoxide. In vitro studies have also been conducted. The oxidation of enoximone to sulfoxide was investigated in rat liver and kidney 9,000 g supernatant fractions (Fig. 4). The reduction of sulfoxide was studied in rat liver and kidney 9,006 g supernatant fractions [Fig. 4). It is clear that oxidative enoximone metabolism and reductive sulfoxide metabolism each occurs in both liver and kidney preparations. In these in vitro studies, the liver was more active than the kidney in oxidation, whereas reduction was more active in the kidney. One may speculate that reversible metabolism also occurs in humans.
directly from enoximone in the kidney, the ratio of sulfoxide rate of excretion to its plasma level would not constitute a true renal clearance.7 This may be the case in humans; it was not uncommon to observe intrasubject sulfoxide (rate of excretion/plasma concentration] ratios fluctuating lo- to 15-fold over the course of a study. Additionally, when reversible metabolism is present, intact drug clearance estimates based solely on dose/area under the plasma concentration-time curve ratios of intact drug will be underestimated.*!9 Since we assume that the reversibility of metabolism observed in rats and dogs also occurs in humans, clearance and volume,of distribution cannot unambiguously be determined unless the presumed reversibly formed metabolite is also administered. Because this could not be accomplished under the present Investigational New Drug Application, these reported parameters are only apparent. Another complicating factor in the pharmacokinetic analysis is the apparent nonlinearity. Protein binding: Preliminary studies on enoximone protein binding using serum from healthy adult men indicate that binding is approximately 85% at therapeutic drug concentrations. Single dose oral study in healthy human subjects: To determine the effect of food on the relative bioavailability of enoximone from a soft gelatin capsule formulation (150 mg], a X-way nonrandomized crossover study was conducted in 12 healthy volunteers. In the first period of the study, fasting subjects received a single enoximone capsule with 200 ml of water at 8 A.M. In the second period of the study [l week later), the same subjects consumed a standard breakfast after an overnight fast; the meal, which was served at 7 A.M.,
Pharmacokinetics If in humans (or any other species), significant amounts of sulfoxide excreted in urine are formed
3 IL--
W+;’
H
No
Rat - trace Dog - 1.3% Monkey - 0.8% Man - trace
NH
ENOXIMONE
/
\
CH5S--@C-0~ j
‘d
i
+ [H3cfxo
j
C&
%$;;i;pa3,
SULFOXIDE
(MDL
Man
19,438)
- 78%
GLYCINE I f,--NHCH+OOH 0 GLYCINE CONJUGATE (4-methyithiohippuric
acid)
Rot - detected Dog - 30% Monkey - 5-10X Man - trace
I
V
H N
C LTLrf
C+-
‘d SULFONE
(MDL
o NH
Rat - 5.5% Dog - trace Monkey - trace Man - trace
19,437)
FIGURE 1. Known metabolic pathways for enoximone (bracketed compounds are postulated intermediates). average urinary recoveries. Rat data were obtained from 24-hour urine collections after intravenous 5 mg/kg doses Sprague Dawley (CD) rats. Dog data were obtained from 24-hour urine collections after intravenous 10 mg/kg doses Monkey data were obtained from 24-hour urine collections after intravenous 15 mg/kg doses to 3 male cynomolgus were obtained from 8-hour urine collections after 2 mg/kg oral solution doses to 8 healthy male subjects who Chemical entities were identified and quantitated using sensitive and specific high performance liquid chromatography raphy-mass spectrometry.
Percentage values are to 5 male Charles River to 3 male beagle dogs. monkeys. Human data had fasted overnight. and gas chromatog-
August
14, 1987
THE AMERICAN
JOURNAL
OF CARDIOLOGY
01-, 30
60 TIME
100,
60
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FIGURE 2. Plasma and whole heart enoximone and sulfoxide concentration-time profiles after intravenous bolus injections of 5 mg/ kg enoximone to male Charles River Sprague Dawley (CD) rats. Two rats each were killed at 5, 15, 30 and 60 minutes: average concentration values were used. A single rat was killed each at 120 and 160 minutes (heart sulfoxide concentrations were undetectable at 160 minutes). Drug and metabolite levels were measured by an accurate and specific high performance liquid chromatographic method.
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FIGURE 3. Plasma and whole heart enoximone and sulfoxide concentration-time profiles after intravenous bolus injections of 5 mg/ kg racemic enoximone sulfoxide to male Charles River Sprague Dawley (CD) rats. Two rats each were killed at 5, 15, 30 and 60 minutes; average concentration values were used. A single rat was killed each at 120 and 160 minutes (concentrations of enoximone and sulfoxlde were undetectable at 160 mlnules). Drug and metabolite levels were measured by an accurate and specific high performance liquid chromatographic method.
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consisted of cereal, skim milk, a banana, toast, jelly, margarine (1 pat) and decaffeinated coffee with sugar. At 8 A.M., the subjects received 150 mg enoximone (capsule) with 200 ml of water. During both periods of the study, lunch was provided at 12 noon. Blood [disodium ethylenediaminetetraacetic acid anticoagulant) was obtained at 0, 0.5, 1, 1.5, 2, 3, 4, 6, 8, 10, 12, 16 and 24 hours. Plasma was separated and frozen until analysis. Urine samples were collected at -12 to 0,O to 22 to 4,4 to 6, 6 to 8, 8 to 10, 10 to 12, 12 to 16 and 16 to 24 hours. Urine volumes were measured, and aliquots were frozen until analysis. Plasma enoximone and sulfoxide concentrations were measured by a specific, sensitive and accurate high performance liquid chromatography procedure. lo A modified procedure was used to determine urine concentrations of sulfoxide. Figure 5 shows mean enoximone and enoximone sulfoxide plasma concentration-time profiles and Table I summarizes mean pharmacokinetic parameter estimates. Statistically significant differences between the nonfasting and fasting conditions were observed (p
60 TIME
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,
FIGURE 4. Enoxlmone sulfoxlde (/err) and enoximone (right) formation after incubation of 5 X 10e6 M (1.24 mg/liter) enoximone with male Charles River rat liver and kidney 9,000 g superna-
120
(MINUTES)
<0.05) for enoximone and sulfoxide peak concentrations but not for time of peak concentration, area under the plasma concentration-time curve (0 to 24 hours] and urinary sulfoxide recovery. Although the power to detect a 20% difference [a = 0.05) due to dietary state for time of peak concentration was low, it was greater than 0.90 for all other parameters that showed no difference. These results indicate that although the presence of food affects the rate of drug absorption (via altering drug absorption and/or hepatic blood flow], overall the extent of bioavailability is not appreciably influenced. Multiple dose oral study in healthy human subjects: In a limited 2-way crossover study in 8 normal male volunteers, the relative bioavailability of enoximone from the soft gelatin capsule formulation was compared with that of a solution formulation (5 mg/ ml]. During the first period of the study, subjects received 150 mg enoximone orally every 8 hours [around the clock] for 22 doses; subjects were randomly allocated to formulations. After a z week washout, the sub-
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TABLE I Pharmacokinetic of Enoxlmone Absorption
IN HEART
FAILURE
Parameter Estimates Under Fasting Conditions
Obtained from a 12 Subject Bioavailability Study and After a Standardized Breakfast (nonfasting) Enoximone
Enoximone Parameter AUC [(rig/ml) . hr] (0 to 24 hours) Peak concentration @g/ml) Time to peak (hr) Cumulative percent urinary recovery (0 to 24 hours)
Fasting 551
f
Nonfasting 169
569
f
Sulfoxide
Fasting 119
81 f36
123 f 2%
2.5 f 2.6 not assayed
1.3 f 0.40 not assayed
4,311 663
Nonfasting
f 491
4,605
f
i 246
1,130
f 230
1.5 f 0.64 75 f 13*
* Data available for 10 subjects because of incomplete urine collections. Data are mean f standard deviation; enoximone was administered as 150 mg in a soft gelatin AUC = area under the plasma concentration-time curve. Mean plasma concentration-time curves are shown in Figure 5.
1,095
1.6 f 0.66 76 f 0.7
capsule.
0 T,ME4vi0& FIGURE 5. Mean enoximone (fop) and enoximone sulfoxide (boftom) plasma concentration-time profiles after oral administration of 150 mg enoximone in a soft gelatin capsule formulation (fasting and nonfasting conditions). The nonfastlng condition denotes that the capsule was ingested after consumption of a standardized breakfast (see text). Mean parameter estimates are summarized in Table I.
jects were crossed over, and the protocol was repeated. The first dose of each day was always administered immediately after breakfast. Solution doses (30 ml of the 5 mg/ml solution) were administered in 4 to 6 ounces of fruit juice; capsules were ingested with 200 ml of water. BIood samples (disodium ethylene diaminetetraacetic acid anticoagulant) were obtained at: (1) 0,0.25,0.5,1,2,3,4,6 and 8 hours after the first dose; (2) immediately before the morning dose on days 2
1’2 1’6 2’0 2’4
FIGURE 6. Mean enoximone and enoximone sulfoxide plasma concentration-time profiles after single and multiple oral enoximone doses (150 mg) to healthy male subjects; two dosage forms were studied, a solution and a soft gelatin capsule. Drug was administered at 8 hour intervals (around the clock) (see text).
through 7; (3) 0, 0.25,0.5, 1, 2,3,4, 6,8, 10, 12, 18 and 24 hours after the last dose (22) on day 8. Urine samples were collected over 8 hour intervals after the first 3 doses on day 1. On days 2 through 7, M-hour urine collections were obtained. On the morning of day 8 [with dose 22), urine was collected for 3 sequential 8 hour periods. Blood was centrifuged and harvested for its plasma; both plasma and urine specimens were treated as previously described. Whereas only sulfoxide was analyzed in urine, both enoximone and sulfox-
August
14, 1987
ide were measured in plasma (the same analytical methods were used as in the single dose, food effect study]. Figure 6 illustrates mean enoximone and enoximone sulfoxide plasma concentration-time profiles. Considerable scatter in the data after the last dose made it impossible to estimate accurately the terminal exponential half-life. Statistical analysis of the data indicated that although there were differences in rate of enoximone absorption between the z formulations, the relative extent of bioavailability did not differ by more than 5% to 1070. What is perhaps most clinically relevant is the accumulation of enoximone and its sulfoxide metabolite. Wagner l1 defined the drug concentration index (R,) of accumulation as:
R, =
average plasma concentration 7 hours at steady-state
during
average plasma concentration from 0 to 7 hours after the first dose where 7 is the dosing interval. Using the data after administration of the solution, the mean drug concentration index was 2.1 for enoximone and 1.5 for sulfoxide. The mean steady-state sulfoxide:enoximone plasma concentration ratio (determined from dose 22 and adjusted for molecular weight) was 3.9. This latter value was considerably less than the mean ratio of sulfoxide:enoximone single-dose areas under the plasma concentration-time curves (0 to 24 hours) observed in fasting subjects in the previously described bioavailability study. The mean value obtained in that study (corrected for molecular weight] was 7.7. Viewed collectively, these data suggest nonlinearity of 1 or more pharmacokinetic processes, which may be saturability of sulfoxidation. Intravenous singie-dose studies in class III and IV congestive heart failure patients: Enoximone was administered by intravenous infusion (2.8 to 16.6 min-
utes] to 6 patients (5 male, 1 female) with severe (class III or IV] CHF. Five patients received 2 different doses, ranging from 0.5 to 3.0 mg/kg. Figure 7 shows enoximone and sulfoxide plasma concentration-time profiles for a representative subject. The terminal exponential half-life for enoximone ranged from 3.0 to 8.1 hours (median 6.2); for the sulfoxide metabolite, the half-life ranged from 6.0 to 11.0 hours (median 7.6). Area under the metabolite curve ranged from 2.9 to 8.3 (median 4.8) times greater than for intact drug (corrected for molecular weight]. Apparent total clearance for enoximone ranged from 3.7 to 13.0 (median 9.61 ml/ min/kg. Apparent steady-state volume of distribution ranged from 2.1 to 8.0 (median 4.2) liters/kg. Over a 24hour period, a median 0.49% of the dose was recovered in urine as intact drug. The median recovery of sulfoxide in a 24 hour urine was 74%. Both intra- and intersubject apparent renal clearance for sulfoxide varied considerably, suggesting enoximone and sulfoxide may have been interconverted by the kidney. In the 5 subjects receiving 2 different doses, the area ratio of sulfoxide to intact drug was greater than the lower dose. The median percent increase was 42%. Although the sample size does not permit extensive sta-
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tistical analysis, these data are consistent with dosedependent kinetics in the sulfoxidation reaction, It was not possible to correlate drug/metabolite plasma concentration to pharmacodynamic response, possibly due in part to the fact that the subjects were receiving concomitant medication (particularly digoxin and diuretics). However, some pharmacodynamic responses (systemic vascular resistance, pulmonary vascular resistance and pulmonary capillary wedge pressure) generally decreased after drug administration, whereas cardiac output generally increased. The magnitude of drug responses, however, differed considerably among subjects. Intravenous infusion study in class III to IV congestiye heart failure patients: To investigate the phar-
macokinetics of enoximone and its sulfoxide metabolite during intravenous infusion, plasma concentrations of enoximone and sulfoxide were determined in 12 male patients with severe CHR (class III to IV] who received enoximone therapy. The endximone infusion rate was initially 90 pg/min/kg body weight; once a desired response was achieved (0.42 to 1.0 hour), the infusion rate was reduced to a maintenance rate. The maintenance infusion rates ranged from 7.4 to 40 pg/min/kg, with a median value of 14 pg/min/kg
3000 2000 F = E G c
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FIGURE 7. Enoximone and enoximone sulfoxide plasma concentration-time profiles in a representative patient with class IV congestive heart faiiure who received a 1.5 mg/kg enoximone dose infused over a period of 8.58 minutes (this patient was also receiving heparin, insulin, digoxin, oxycodone and acetaminophen). The so/id and dashed lines are theoretical curves generated from the 4exponential enoximone and sulfoxide plasma concentration-time functions, which were fitted to the postinfusion data; each value of the dependent variable was weighted by the reciprocal of the observed plasma concentration squared.
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A SYMPOSIUM:
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FAILURE
data suggests that nonlinear pharmacokinetic processes may have been operative. In summary, the data presented demonstrate that enoximone is readily absorbed and undergoes firstpass metabolism to an active sulfoxide whose formation is saturable. Further, its metabolites are cleared by urinary excretion.
References
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FIGURE 8. Plasma concentration-time profiles of enoximone and enoximone sulfoxide in a representative patient with class IV congestive heart failure who received a loading enoximone infusion of 5.1 mg/min from 0 to 0.68 hours and a maintenance infusion of 2.3 mg/min from 0.77 to 45 hours.
(there was a 0.083 hour lag between the loading and maintenance infusion). The maintenance infusion lasted 29 to 48 hours. Figure 8 illustrates representative enoximone and enoximone sulfoxide plasma concentration-time profiles in 1 patient. It should be noted that although the plasma concentrations of enoximone and its sulfoxide metabolite did not reach steady-state in all patients [the reason for this is currently unknown, but may have been associated with the severity of disease], others did appear to reach steady-state within the first 24 hours of infusion. Nonetheless, the fact that steady-state was not achieved in all of the patients within the time predicted from single dose
1. Dage RC, Roebel LE, Hsieh CP, Weiner DL, Woodward JK. Cardiovascular properties of a new cardiotonic agent: MDL 17,043 (1,3-dihydro-4-methy&[4-(methylthio)-benzoyIJ-2H-imidazol-2-one]. J Cardiovasc Pharmacol 1982; 4:500-508. 2. Roebel LE, Dage RC, Cheng HC, Woodward JK. Characterization of the cardiovascular activities of a new cardiotonic agent MDL 17,043 (1,3-dihydro4-methyI-6-[4-(methyIthio)-benzoy~-2H-imidazoI-2-one), J Cardiovasc Pharmacol 1982;4:721-729. 3. Arbogast R, Brandt C, Haegle KD, Fincker JL, Schechter PJ. Hemodynamic effects of MDL 17,043, a new cardiotonic agent, in patients with congestive heart failure: comparison with sodium nitroprusside. J Cardiovasc Pharmacol 1983;5:998-1004. 4. Crawford MH, Richards KL, Sodums MT, Kennedy GT. Positive inotropic and vasodilator effects of MDL 17,043 in patients with reduced left ventricular performance. Am J Cardiol 1984;53:1051-1053. 5. Amin DK, Shah PK, H&e S, Shellock F. Comparative acute hemodynamic effects of intravenous sodium nitroprusside and MDL 17,043, a new inotropic drug with vasodilator effects, in refractory congestive heart failure. Am Heart J 1985;109:1006-1012. 6. Dage RC, Kariya T, Hsieh CP, Roebel LA, Cheng HS, Schnettler RA, Grisar JM. Pharmacology of enoximone. Am J Cardiol 1987;68:18C-14C. 7. Wan SH, Riegelman S. Renal contribution to overall metabolism of drugs. I: conversion of benzoic acid to hippuric acid. J Pharm Sci 1972;61:1278-1284. 8. Hwang S, Kwan KC, Albert KS. A linear model of reversible metabolism and its application to bioavailability assessment. [ Pharmacokinet Biopharm 1981;9:693-709. 9. Wagner JG, DiSanto AR, Gillespie WR, Albert KS. Reversible metabolism and pharmacokinetics: application to prednisone-prednisolone. Res Comm Chem Pathol Pharmacol 1981;32:387-406. 10. Chan KY, Ohlweiler DF, Lang JF, Okerholm RA. Simultaneous analysis of a new cardiotonic agent, MDL 17,043, and its major sulfoxide metabolite in plasma by high-performance liquid chromatography. J Chromatogr 1984;306: 249-256. 11. Wagner JG. Drug accumulation. 1 Clin Pharmacol 1967;7:84-88.