ANTI-ATHEROSCLEROSIS

Pharmacokinetics Single and in Normal Francis

L. S. Tse,

PhD,

FCP,

AND

of Fluvastatin Multiple Doses Volunteers

James

M. Jaffe,

PhD,

and

August

HYPERLIPIDEMIA

After

Troendle,

MD

The

pharmacokinetics of fluvastatin, a potent inhibitor of hydroxymethylglutaryl-CoA and thus cholesterol synthesis, have been studied in 24 normal male volunteers who received [3H] fluvastatin in three different studies: a single-dose study using oral doses of 2 or 10 mg, an absolute bioavailability study using doses of 2 mg intravenously or reductase

10 mg

orally,

and

a multiple-dose

study

using

40 mg

orally

once

daily

for

6 days.

Serial

blood

and plasma samples and complete urine and feces were collected and analyzed for total radioactivity as well as for intact fluvastatin. Fluvastatin was rapidly and almost completely (>90%) absorbed from the gastrointestinal tract, although the estimated bioavailability from the 2- and 10-mg doses was only 19 to 29% because of extensive firstpass metabolism. Fluvastatin pharmacokinetics appeared to be linear over the 2- to 10mg dose range, as indicated by dose-proportional blood levels of total radioactivity and the parent drug. Absorbed jluvastatin was completely metabolized before excretion, the biliary/fecal route being the major excretory pathway. The recovery of radioactivity after a single dose was virtually complete within 120 hours. The terminal half-lives of fluvastatin and total radioactivity averaged 0.5 to 1 hour and 55 to 71 hours, respectively, whereas the total body clearance of fluvastatin was 0.97 L/hour/kg. Repeated oral administration of 40-mg doses of [3H]fluvastatin resulted in no time-related change in pharmacokinetic characteristics, but this dose yielded greater than proportional increases in circulating levels of the parent drug, thus suggesting a saturable .first-pass effect on fluvastatin. During repeated daily administration, plasma levels of fluvastatin reached steady state after the first dose, whereas those of total radioactivity approached steady state after 6 days. The degree of accumulation of radioactivity, unlike that of the parent drug, was inconsistent with the terminal half-life, but instead implied a shorter effective half-life of 32 to 36 hours. It appears that the terminal phase of the blood radioactivity profile represents a minor metabolite that is reversibly bound to and slowly released from a specific tissue depot, and that this binding involves a finite amount of drug regardless of the dose administered. The oral and intravenous administration of [3Hjfluvastatin described in the present report was safe and well tolerated.

F luvastatin

(Sandoz compound XU 62-320), [R*, S*(E)](±)sodium 3,5 -dihydroxy7-[3-(4fluorophenyl) -1- (1- methylethyl) 1H indole -2 -yl] hept-6-enoate, is a potent inhibitor of hydroxymethylglutaryl coenzyme A (HMG-C0A) reductase, the rate-limiting enzyme in cholesterol biosynthesis.1.2 It is more potent than compactin and lovastatin in inhibiting HMG-CoA reductase in vitro. Previous studies have shown significant reduction in serum -

-

-

From the Department of Drug Metabolism (Drs. Tse and Jaffe) and the Cardiovascular Department (Dr. Troendle), Sandoz Research Institute, East Hanover, New Jersey. Address for reprints: Francis L.S. Tse, PhD, Sandoz Research Institute, East Hanover, NJ 07936.

630

#{149} J CIIn Pharmacol

1992;32:630-638

total cholesterol, low-density lipoprotein cholesterol, and serum triglyceride levels in rats, dogs, and monkeys treated with fiuvastatin.3 Lowering plasma levels of low-density lipoprotein cholesterol while increasing high-density lipoprotein cholesterol levels reduces the risk of coronary heart disease and atherosclerosis.4’5 The absorption and disposition of fluvastatin have been studied in the mouse, rat, dog, and monkey using 14C- or 3H-labeled drug.6 Oral doses of the drug were absorbed at a moderate to rapid rate, the extent of absorption being dose independent and essentially complete in all species. Fluvastatin was subject to extensive presystemic hepatic extraction, however,

PHARMACOKINETICS

followed by direct excretion in the bile, thus yielding high liver/peripheral tissue concentration gradients of drug-related materials. The bioavailability of the parent drug increased with dose, apparently because of saturation of the first-pass effect. Dose-normalized blood levels of fluvastatin and total radioactivity were higher in the dog than in the other species. Fluvastatin was partially metabolized before excretion, the extent of metabolism being smallest in the dog and greatest in the mouse. The half-life of intact fluvastatin ranged from I to 2 hours in the monkey to 4 to 7 hours in the dog. Regardless of the dose or dose route, the administered radioactivity was recovered predominantly in feces, with the renal route accounting for less than 8% of the dose. No tissue retention of radioactivity was observed, and material balance was essentially achieved within 96 hours after dosing. The present report concerns the pharmacokinetics of fluvastatin in healthy humans who received single and multiple oral doses of [3H]fluvastatin. Additionally, a single intravenous dose of [3H]fluvastatin as well as an oral dose of nonradiolabeled fluvastatin were used to assess the absorption and absolute bioavailability of the drug.

OF

FLUVASTATIN

Radiochemical purity was greater than 95% as determined by radio-thin-layer chromatography and by high-performance liquid chromatography-radioactivity monitoring. Three oral solutions were prepared, 2 mg/b mL (0.5 mCi/mg) and 10 mg/b mL (0.1 mCi/mg) for the single-dose study and 40 mg/40 mL (4.3 iCi/mg) for the multiple-dose study. An injectable solution, 2 mg/2 mL (0.47 mCi/mg), and a nonradiolabeled oral solution, 10 mg/b mL, were used in the absolute bioavailability study. Study

Design

The 12 subjects in the single-dose study were assigned to one of two groups of equal size. Thus, six subjects received a 2-mg oral dose and the other six received a 10-mg oral dose of [3H]fluvastatin. In the absolute bioavailability study, six subjects each received a 2-mg intravenous dose of [3H]fluvastatin followed 8 days later by a 10-mg oral dose of nonradiolabeled fluvastatin. In the multiple-dose study, six subjects each received 40 mg of [3H]fluvastatin orally once daily for 6 days. The medication in each study was administered on an open-label basis.

Dosing

and Sample

Collection

METHODS Subjects Twenty-four healthy male volunteers were used. Twelve (aged 18 to 32 years; weight, 61 to 87 kg) participated in the single-dose study; six (aged 22 to 44 years; weight, 56 to 91 kg), in the multiple-dose study; and six (aged 20 to 35 years; weight, 59 to 91 kg), in the absolute bioavailability study. All subjects gave written informed consent after protocol approval by the local Institutional Review Board. Vital signs and electrocardiograms were within normal limits, and blood and urine laboratory values were within 10% of the normal range based on an initial physical examination. The subjects received no radioactivity in any form within 1 year of the study, or any concomitant medication during the study. In each study, a careful history was obtained to exclude, as much as possible, subjects with alcohol abuse or other liver diseases. In addition, a hepatitis profile was obtained to exclude those subjects with previous hepatitis A or B infection. A urinary drug screen was performed in all subjects before testing. Study

Medication

[3H]Fluvastatin was bel at the 3 position

ANTI-ATHEROSCLEROSIS

synthesized with the of the p-fluorophenyl

AND

HYPERLIPIDEMIA

tritium lamoiety.

In the single-dose study, all subjects fasted for 8 hours before and 2 hours after dosing, but had free access to water. At 8:00 A.M. on the treatment day, each subject received 2 mg or 10 mg [3H]fluvastatin by ingesting the appropriate drug solution. Venous blood samples were collected immediately before and at 0.5, 1, 2, 3,4, 6, 9, 12, 24, 36, 48, 72, 96, and 120 hours after drug administration. Quantitative urine and feces were collected at 24-hour intervals for 5 days. In the absolute bioavailability study, the subjects fasted for 8 hours before and 2 hours after each dosing, but were allowed water at all times. At 8:00 AM on the first treatment day, each subject received a 2-mg dose [3H]fluvastatin as a 20-minute infusion into a forearm vein. Venous blood was collected at 0(predose), 5, 10 and 20 minutes during the infusion and at 0.083, 0.25,0.5, 1, 2, 3,4, 6, 9, 12, 24, 36,48, 72, 96, and 120 hours after the end of infusion. Quantitative urine was collected at 24-hour intervals for 5 days. Exactly 8 days after the intravenous dose, each subject received 10 mg nonradiolabeled fluvastatin as a drink solution. Blood was collected at 0 (predose), 0.25, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 6, 8, and 12 hours postdose. During multiple dosing, the subjects fasted for 8 hours before and 2 hours after each drug administration. Water was allowed as desired. Each subject re-

631

TSE,

IAF’FE,

ceived 40 mg [3H}fluvastatin in solution form at 8:00 every day for six consecutive doses. Blood samples were collected before and at 24, 48, 72, 96, 120, 120.25, 120.5, 120.75, 121, 121.5, 122, 122.5, 123, 124, 126, 128, 132, 144, 156, 168, 192, 216, and 240 hours after the first dose. An aliquot of the blood was immediately centrifuged to separate the plasma. Quantitative urine and feces were obtained at 24-hour intervals for 10 days after the initial dose. All samples were stored frozen (-15#{176}C)until analysis, which began within 3 weeks of collection. AM

Analysis

of Radioactivity

Radioactivity was measured in a liquid scintillation spectrometer (Model 460, Packard Instrument Co., Downers Grove, IL). Aliquots of blood, plasma, and homogenized feces were air dried and combusted in a sample oxidizer (Model 306, Packard). Urine samples were assayed by directly counting aliquots in ACS#{174}scintillant (Amersham Corp., Arlington Heights, IL). Determination

of Tritiated

of Fluvastatin

Blood and plasma concentrations of fluvastatin were determined by high-pressure liquid chromatography with fluorescence detection as previously described.6 In the multiple-dose study, the method was modified to include the use of an internal standard, which differs from fluvastatin only by having a methyl group at the 6-position of the heptenoic acid chain.9 The lower limit of quantification was 0.5 ng/ mL for the original method and 1 ng/mL using the modified procedure. Using high-pressure liquid chromatography with radioactivity monitoring, fluvastatin also was determined in selected urinary and fecal samples.

Pharmacokinetic

TROENDLE

lated by the trapezoidal rule. After evaluation of the extent of total absorption, the first-pass effect was estimated using the equation of Gibaldi et al.1#{176} f -

and Vd

#{149} J Clin Pharmacol

1992;32:630-638

-

Dose (AUG)2 (AUMG)

where AUMC is the area under the first moment of the concentration curve and was obtained by the trapezoidal rule.11 In the multiple-dosing study, steady-state conditions were established by examining the predose blood levels (C,,,,.J and the urinary excretion during each dose interval. The parameters Cmax, tmax, and terminal half-life were determined as described above, but AUG was calculated for only the last dose-interval. The effective half-life,defined as that half-life consistent with observed accumulation,12 was estimated from the ratio of the Gmjn at steady state (C1) to that after the first dose In the following equation, Accumulation

C

ratio

=

1 =

-

C1

1

-

mm

w equals 0.693 divided by the effective half-life, is the dose interval. This equation can be rearranged to the following form, =

632

Dose AUG

CL

Interpretation

Single-dose blood data were analyzed in terms of peak concentration (CmaJ, time to peak (tma,J, and area under concentration-time curve (AUC) calcu-

Q Q + (F . D/AUC)

where F is the fraction of dose D absorbed, f is the fraction of the absorbed dose that reaches the systemic circulation intact, and Q is the hepatic blood flow rate, 1.7 L/min. Elimination half-lives of fluvastatin and total radioactivity were estimated by linear regression analysis of the terminal log-linear phase of the concentration-time curves. In the absolute-bioavailability study, the AUG values of unchanged drug obtained after oral and intravenous dosing in the same subjects were used to assess the bioavailability of fiuvastatin. Terminal halflives were determined as described above. The total body clearance (CL) and steady-state volume of distribution (VdJ of fluvastatin were calculated using blood level data after intravenous dosing as follows:

Water

The amount of tritiated water formed by biotransformation of [3H]fluvastatin was determined by measuring the specific activity (dpm/mL) of the distil. lates of urine samples from selected time intervals.7 These values were extrapolated to time zero and the body contents of tritiated water calculated based on known values of tritiated water half-life (227 hours) and volume of exchangeable body water (55% body weight) in humans.8

Analysis

AND

-ln(l

r

-

T

from which culated.

the

effective

half-life

can

be readily

cal-

PHARMACOKINETICS

OF

RESULTS

tal blood radioactivity level. Throughout the sampling period, the concentrations obtained after the high dose were approximately 4 times the corresponding values from the low dose. Because the doses, normalized by body weight, differed by approximately fourfold (Table I), these results indicated a linear dose-concentration relationship in the dose range examined. The elimination half-lives of radioactivity and fluvastatin were 55 to 71 hours and 0.5 hour, respectively. The parent drug concentrations declined rapidly to below quantification limit after 3 to 4 hours. This observation led to increased sampling frequency during the early post-oral dose hours in the design of the subsequent absolute bioavailability study. As shown in Table II, the excretion pattern after a single oral dose was independent of the dose level in the 2- to 10-mg range, with about 5% of the administered radioactivity excreted in urine and the remainder recovered in feces. The total recovery within the collection period was greater than 90% of the dose. In both dose groups, intact fluvastatin accounted for 1.8% of the total radioactivity recovered in feces and -

7

2

uu.uu O

-‘-4-’-

93 ± 3.2

the first and last doses

dose was unaltered, as indicated by virtually identical, dose-normalized AUG values of radioactivity as well as the overall excretion patterns in the singleand multiple-dose studies. The terminal half-lives of total radioactivity and fluvastatin after repeated 40mg doses were also similar to the respective values

t5>/

-

Feces radioactIvIty

Urine radioactivity

Total recovery, 0-240

-

were substituted

as C,

and C,,

respectively.

after a single 2- or 10-mg dose, despite indications of saturable first-pass metabolism as discussed above. Changes in the extent of first-pass metabolism without a significant effect on subsequent drug disappearance rate has been reported for drugs subject to a high hepatic, flow-limited clearance.13 However, the degree of accumulation of radioactivity in the present study indicated an effective half-life of 32 to 36 hours, which is consistent with the time to reach steady state (6 days) but considerably shorter than the half-life calculated by using the slope of the terminal phase. It is postulated that the terminal phase of the blood radioactivity profile represents a minor metabolite that is reversibly bound to and slowly released from a specific tissue depot, and that this binding involves a finite amount of drug regardless of the dose administered. Thus, the terminal phase would effectively have a “one-time” contribution to accumulation, contrary to the additive contribution by successive doses in a linear process.12 This hypothesis is supported by the essentially complete recovery of administered radioactivity within 120 hours after the final dose. The clinical portion of this study was conducted under the supervision of Drs. Marvin Fahrenbach, Albert Cohen, and Miguel Zinny. The authors thank Ms. Gaetana Kalafsky and Mr. Joseph Nicoletti for technical assistance.

-

REFERENCES Dose 1.

Figure 2. Mean urinary excretion of radioactivity in each dose interval during multiple oral administration (40 mg qd X 6) of [3H]fluvastatin to 6 men. Bars indicate I SD.

ANTI-ATHEROSCLEROSIS

AND

HYPERLIPIDEMIA

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TROENDLE

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Langham water

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Comparative

J Cell

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availability

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pharmacokinetics in drug discovery and PG, Tse FLS (eds.): Pharmacokinetics: Perspectives. New York: Marcel

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Clin