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Research ArticleArticle

Metabolism and Excretion Studies in Mouse After Single and Multiple Oral Doses of the 3-Hydroxy-3-methylglutaryl-CoA Reductase Inhibitor Atorvastatin

Ann E. Black, Michael W. Sinz, Roger N. Hayes and Thomas F. Woolf
Drug Metabolism and Disposition August 1998, 26 (8) 755-763;
Ann E. Black
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Michael W. Sinz
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Roger N. Hayes
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Thomas F. Woolf
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Abstract

Atorvastatin, [(R-(R,R)]-2-(4-fluorophenyl)-β,δ-dihydroxy-5-(1-methylethyl)-3-phenyl-4-[(phenyl-amino)carbonyl]-1H-pyrrole-1-heptanoic acid calcium salt (CI-981, AT), is a second generation 3-hydroxy-3-methylglutaryl-CoA reductase inhibitor approved for clinical use as a cholesterol lowering agent. The disposition and metabolism of AT, including potential CYP450 induction, was investigated in mice administered an oral dose of [14C]AT (free acid) on study days 1 and 14. Peak plasma radioactivity concentrations occurred 1 hr postdose after both single- and multiple-dose administration and declined rapidly thereafter. Total plasma radioactivity levels in mice receiving the multiple dose were approximately 50% of levels observed after single-dose administration. Plasma metabolic profiles, which provided evidence of extensive metabolism, remained similar. Feces was the major route of AT-derived radioactivity elimination. Fecal profiles showed extensive metabolism with qualitatively similar profiles after single- and multiple-dose administration; however, quantitative differences were apparent. Metabolites identified in plasma and feces include hydroxylated, β-oxidized, and unsaturated derivatives of AT. Most metabolites had undergone β-oxidation. In mice receiving multiple 1 mg/kg doses of AT, no effect on spectral P450 concentration was found, and only a minor increase was observed at the 200 mg/kg dose level. Catalytic activities of CYP4501A, -2B, and -3A were not significantly affected; CYP4A activity decreased in a dose-dependent manner. Administration of multiple doses resulted in lower systemic plasma levels of total AT-derived radioactivity not readily explained by these studies. In mice, the majority of metabolites are formed primarily through the β-oxidation pathway.

Atorvastatin, [R-(R,R)]-2-(4-fluorophenyl)-β,δ-dihydroxy-5-(1-methylethyl)-3-phenyl-4-[(phenylamino)carbonyl]-1H-pyrrole-1-heptanoic acid calcium salt (CI-981, AT1) is a potent synthetic inhibitor of 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase, the rate-limiting enzyme in cholesterol biosynthesis. Recently approved for human use, AT has a significant cholesterol lowering effect (Nawrocki et al., 1995). As part of AT development, absorption and disposition studies were conducted in rats and dogs. No apparent metabolic differences were observed between the two species; extensive presystemic metabolism with biliary excretion was the predominate route of radioactivity excretion (Michniewicz et al., 1994). Metabolites identified include ortho- andpara-hydroxy-AT, β-oxidized products, and a glucuronide conjugate of ortho-hydroxy-AT. Toxicokinetic investigations in mice and rats after repeated AT dose administration did not produce equivalent plasma concentrations, as measured by a nonspecific HMG-CoA inhibition assay (Black et al., 1995; Shum et al., 1996). In the mouse, daily doses ranging from 10 to 400 mg/kg AT produced a marked effect on plasma AT activity equivalent concentrations. Cmax and AUC values were higher on day 1 than day 14 in mice, a result not observed in rats. The present studies were conducted to characterize the metabolism and excretion of AT in mice after single- and multiple-dose administration. In addition, we investigated the effect of multiple-dose administration on hepatic CYP450.

Materials and Methods

Chemicals. Radiolabeled atorvastatin.

[14C]AT (specific activity, 81.2 mCi/mmol, chemical and radiochemical purity >98%) was synthesized with the carbon label at the 3-position of the pyrrole moiety. Ring-labeled [14C] was synthesized using an adaptation of the synthetic methodology of Baumann et al. (1992). The label was introduced as [14C]benzaldehyde, followed by sequential condensation with isobutyrylacetanilide, and the product was reacted with para-fluorobenzaldehyde to form a diketone intermediate. The diketone was conjugated with the protected chiral dihydroxyaminoheptanoic ester (Roth, 1987), and the product was treated with acid and base prior to calcium salt formation. Using a similar reaction sequence, ortho- andpara-hydroxy-AT were synthesized and purified as sodium salts (Bjorge et al., 1995; Roth et al., 1991). Testosterone and [14C]lauric acid were obtained from Sigma. All other chemicals were reagent grade or better.

Animal Metabolism Studies. Mass balance and metabolic profile.

Male B6C3F1 mice (N = 120, 9–14 weeks of age, 22–34 g) were divided into two groups for plasma profiling or mass balance studies. These groups were further subdivided into mice administered either a single 200 mg/kg [14C]AT (free acid) dose or a single daily dose of 200 mg/kg AT (free acid) for 13 days followed by a final dose of 200 mg/kg [14C]AT. All doses were administered by gavage. Radiolabeled doses were prepared by dissolving labeled and unlabeled material in dimethylacetamide (less than 10% of final volume) followed by the addition of 0.5% methylcellulose to produce a uniform suspension. Each mouse received approximately 10 μCi. Unlabeled AT was prepared as a suspension in 0.5% methylcellulose. Heparinized blood samples (1.0 ml) were collected by cardiac puncture from anesthetized mice, five to seven animals per time point, at predose, 1, 2, 4, 8, and 12 hr postdose for study days 1 and 14. The harvested plasma at each collection time was pooled and stored frozen until analysis. A control group of mice received no drug or vehicle. Mass balance studies used 15 animals per dose. Animals were housed in metabolism cages (five animals per cage) with access to food and water ad libitum. Urine and feces were collected predose and at 24-hr intervals for 6 days. After each radioactive dose, urine and feces from the same collection intervals were pooled. All samples were stored frozen at −20°C until analysis.

Enzyme Induction Study.

Male B6C3F1 mice (N = 21, 9–14 weeks of age, 22–34 g) were divided into three groups: one group received daily single 1.0 mg/kg (free acid) oral doses of AT for 14 days, the second group received daily single 200 mg/kg (free acid) oral doses of AT for 14 days, and the third group served as a control and received daily single oral doses of vehicle (0.5% methylcellulose) for 14 days. Food and water were provided ad libitum. On day 15, animals were sacrificed, and the livers were removed and frozen in liquid nitrogen. Microsomes were isolated from pooled livers from each of the treatment groups (N = 7/group) (Guengerich, 1989). Cytochrome P450 and protein concentrations were determined by the methods of Omura and Bradford, respectively (Omura and Sato, 1964; Bradford, 1976).

Catalytic Assays.

The catalytic activities of CYP1A, -2B, -3A, and -4A were measured with ethoxyresorufin (ER), pentoxyresorufin (PR), testosterone, and lauric acid, respectively. ER and PR dealkylase activity, expressed as pmol of resorufin/min/mg of microsomal protein, were determined by the method of Burke et al. (1985). Briefly, liver microsomes (0.5 mg/ml) from each group were incubated in duplicate in the presence of ER or PR, and the rate of resorufin formation was measured by fluorescence. The activity of CYP3A was measured by determining the rate of testosterone 6β-hydroxylation. Liver microsomes (0.5 mg/ml) were incubated in triplicate with 200 μM testosterone (0.4 μCi) and 1.0 mM NADPH in 50 mM potassium phosphate buffer, pH 7.4, for 10 min. Sample aliquots were extracted with 2 × 5.0 ml of dichloromethane. The organic fractions were dried under nitrogen, and the samples were reconstituted in 50 μl of methanol. Testosterone and 6β-hydroxytestosterone were separated on a Waters Spherisorb C-18 (250 × 4.6 mm) column coupled to a Packard Radiomatic flow detector CR (Downers Grove, IL). The mobile phase consisted of methanol:water (31:19, v/v) at a flow rate of 1.0 ml/min at 50°C. The activity of CYP4A was measured by determining the rate of lauric acid disappearance. Liver microsomes (0.5 mg/ml) were incubated in duplicate with 100 μM lauric acid (1.3 μCi) and 1.0 mM NADPH in 50 mM Tris-HCl buffer, pH 7.4, for 10 min. Sample aliquots were quenched with 300 μl of 3 N HCl and extracted with 8 ml of diethyl ether. Organic fractions were removed and dried under nitrogen; the samples were reconstituted in 60 μl of methanol. Samples were analyzed using the HPLC-radiochromatographic system mentioned above. The mobile phase consisted of 37:63 methanol:methanolic mixture (57.5/42/0.5, methanol/water/acetic acid) at a flow rate of 1.0 ml/min.

Radioactivity Assay.

Radioactivity was measured by liquid scintillation counting (LCS) with external standardization for quench correction. Duplicate aliquots of plasma and urine were counted in 16 ml of Ready Safe (Beckman, Fullerton, CA) by a Packard TR2500 liquid scintillation spectrometer. After addition of water (9:1, v/w), fecal samples were homogenized, and duplicate 0.5-ml aliquots were removed for oxidation. Samples were air-dried and combusted in a Tri-Carb Sample Oxidizer (model 306, Packard) prior to LSC.

Sample Preparation and HPLC Radioactivity Profiles.

Plasma samples (1.0 ml, 1 hr) and fecal homogenates (2.0 ml, 24 hr) were extracted with four volumes of acetone:acetonitrile (1:19, v/v). After centrifugation, the organic layer was placed in another tube and evaporated with nitrogen at room temperature. Residues were reconstituted in 50% acetonitrile:0.1 M ammonium acetate, pH 4, for radioactivity profiling. Recovery averaged 90%. Plasma and fecal extracts were profiled by gradient HPLC with radioactivity detection using a BioSil ODS-5S column (150 × 4.6 mm) (Hercules, CA) connected in a series with a Brownlee RP Spheri-5 guard cartridge (San Jose, CA). The mobile phase consisted of A (0.1 M ammonium acetate, pH 4.0) and B (acetonitrile). The gradient had an initial solvent composition of 75% A and 25% B that was held for 5 min. Over a 40-min period, solvent B increased to 32%, followed by another increase of solvent B to 40% for another 20 min. This mixture was held until 75 min into the run, and then solvent B increased to 80% over the next 15 min. This mixture was held until 110 min was reached (Michniewiczet al., 1994). Radioactivity was profiled with a Radiomatic Flo-One beta radioactivity detector (model CR, Packard) after the addition of Flo-Scint III (Packard) at 3 ml/min. Recovery of radioactivity applied to the HPLC column was 100%.

LC/MS Analysis.

Two systems were used for separation and identification of radioactive components.

System 1.

LC/MS with on-line radioactivity detection was performed using a Hewlett-Packard 1090L HPLC system (Atlanta, GA) in series with a Raytest Ramona 92 radioactivity detector (Pittsburgh, PA) coupled to a Fisons Quattro II triple quadrupole mass spectrometer (Manchester, UK). Chromatographic conditions were identical to those used in profiling, and samples were introduced using atmospheric pressure chemical ionization (APcI) in the positive and negative ion modes.

System 2.

LC/MS was performed using an Isco DM 100 gradient system coupled to a VG Autospec Ultima-Q hybrid mass spectrometer (Manchester, UK). Sample were introduced using either APcI or electrospray ionization (ESI). Increasing the cone voltage from 20 V to 40 V resulted in source-induced fragmentation. A column identical to that above was used to separate analytes; however, the mobile phase consisted of acetonitrile (A) and water with 0.1% (v/v) glacial acetic acid (B). Components were eluted over 30 min using a linear gradient with A starting at 30% and ending at 90%. Analytes eluted at a flow rate of 1 ml/min (APcI) or 0.2 ml/min (ESI).

Data Analysis.

Cumulative recovery of radioactivity in urine and feces is reported as per cent of administered dose. Plasma radioactivity concentrations are expressed as nanogram equivalents of AT free acid/ml. The per cent changes in total CYP450 and resorufin formation from ethoxy- and pentoxyresorufin are compared with values obtained from vehicle-treated control groups. The formation of 6β-hydroxytestosterone from testosterone and the disappearance of lauric acid were also compared with those of vehicle-treated control groups.

Results

Plasma Radioactivity and Metabolite Identification.

After oral administration of [14C]AT on days 1 and 14, radioactivity reached maximum concentrations at 1 hr and then declined rapidly (table 1). Maximum achieved plasma radioactivity concentrations were considerably lower after multiple-dose administration compared with single dose. The total plasma radioactivity AUC(0–12) value of day 1 decreased by 45% after multiple-dose administration.

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Table 1

Plasma AT equivalent concentrations in mice after oral administration of 200 mg/kg of [14C]AT on days 1 and 14

HPLC radioactivity chromatograms (fig. 1) of 1-hr plasma extracts indicated that in both treatment groups most plasma radioactivity was associated with metabolites (table2). The plasma radioactivity profiles were similar for both groups, with the major component eluting at 88.7 min; however, two late eluting peaks from day 14 plasma samples were not detected in day 1 plasma. Plasma radioactivity concentrations beyond the 1-hr sample were too low to provide meaningful information and so were not profiled. The collision-induced dissociation (CID, includes APcI and ESI) MS/MS spectra show characteristic and structurally informative amide bond fragmentation in the positive mode or loss of heptanoic acid side chain in the negative ion mode. Similar structural information was derived from full scan (MS) data by source-induced fragmentation as the cone voltage was increased. The consistent loss of either fragment of the molecule facilitated structure elucidation, as shown by AT (fig.2), which was identified as the peak eluting at 71 min. CID fragmentation of the protonated molecular ion produced ions of m/z 440 and 446; in the negative ion mode, the deprotonated molecular ion for AT produced ions of m/z397 and 453. The major component eluting at 88.7 min was identified as a β-oxidized metabolite of hydroxy AT, of m/z 515 [M + H]+ and m/z 513 [M − H]−. CID of the protonated molecular ion produced ions of m/z 380 and 406, consistent with amide bond cleavage (fig. 3). Moreover, the deprotonated molecular ion underwent CID to give an ion ofm/z 413, consistent with the loss of the pentanoic acid side chain (fig. 3). Minor components were identified at retention times of 87.3, 88.5, and 89.9 min. The positive ion mass spectra of these minor metabolites showed prominent protonated molecular ions ofm/z 513, 497, and 499; the negative ion mass spectra showed prominent deprotonated molecular ions of m/z 511, 495, and 497, respectively. Comparison with synthetic reference material identified the component eluting at 89.9 min as a β-oxidized metabolite of AT. The remaining two components eluting at 87.3 and 88.5 min were tentatively identified as unsaturated analogs of the β-oxidized metabolites. The molecular ions of each were shifted 2 mass units lower, and the fragment ions similarly were 2 amu lower (see fig. 4 for component eluting at 88.5 min). Because the fragment ions were shifted in the positive and negative ion modes, it can be inferred that the site of unsaturation lies in the portion of the molecule retaining the charge. The most likely site of unsaturation is the isopropyl side chain.

Figure 1
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Figure 1

Gradient HPLC-radioactivity chromatogram of a 1-hr plasma sample.

A, profile of plasma on day 1. B, profile of plasma on day 14.

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Table 2

Major radioactive components expressed as percent of radioactivity chromatogram in 1 hr mouse plasma extract after a 200 mg/kg oral dose of [14C]AT on days 1 and 14

Figure 2
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Figure 2

CID mass spectra of AT eluting at 71 min.

A, product ion spectrum of m/z 559 [M+H]+. B, product ion spectrum ofm/z 557 [M-H]−.

Figure 3
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Figure 3

CID mass spectra of a mouse plasma component eluting at 88.7 min.

A, product ion spectrum of m/z 515 [M+H]+. B, product ion spectrum ofm/z 513 [M-H]−.

Figure 4
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Figure 4

ESI of a mouse plasma component with an LC retention time of 88.5 min.

A, positive ion mass spectrum of m/z 497 [M+H]+. B, negative ion mass spectrum ofm/z 495 [M-H]−.

Urine and Feces.

The per cent of radioactivity recovered from urine and feces after administration of [14C]AT (table3) shows feces to be the major route of excretion, with 98% of dose recovered within 48 hr. Under steady-state conditions, recovery of a higher percentage of the administered radioactivity was expected in feces within the first 24 hr compared with fecal recovery after a single dose of [14C]AT. Radioactive components were detected in day 1 and day 14 fecal extracts (table4); however, the per cent of each component varied. Fecal radioactivity profiles were similar between treatment groups with most of radioactivity associated with metabolites. Many additional metabolite peaks present in feces were not found in plasma. Unchanged drug contributed less than 2% of the radioactivity after either treatment, indicating good absorption of AT.

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Table 3

Urinary and fecal recovery in mice after oral administration of 200 mg/kg dose of [14C]AT on days 1 and 14

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Table 4

Major radioactive components expressed as percent of radioactivity chromatogram in 0–24-hr mouse fecal extracts after a 200 mg/kg oral dose of [14C]AT on days 1 and 14 of a multiple-dose study

After source-induced fragmentation of the fecal metabolites, the CID positive ion spectra show characteristic and structurally informative amide bond fragmentation. These components eluted at retention times of 33.5, 63.6, 70, 87.7, and 89.9 min. Mass spectral data are summarized in table 5. Additional components were tentatively identified as unsaturated analogs of aromatic hydroxylated AT (retention times of 50.4 and 63.4 min) and AT (68.5 min). The positive and negative ion mass spectra of each component showed prominent molecular ions that were shifted lower by 2 mass units from the saturated analogs. For example, the positive ion mass spectrum of the component of 68.5 min shows an intense ion of m/z 573 (fig. 5) and source-induced fragment ions of m/z 438 and 464 resulting from amide bond cleavage. Similarly, the negative ion mass spectrum shows a deprotonated molecular ion of m/z 571 (fig. 5) and a fragment ion ofm/z 411 corresponding to the loss of the heptanoic acid side chain. An additional component was identified as β-oxidized bis-hydroxy AT with a retention time of 83.5 min. The positive ion mass spectrum (fig. 6) shows a protonated molecular ion of m/z 531 and a fragment ion ofm/z 396 consistent with amide bond cleavage with loss of an amino-phenol. Moreover, the parent and product ions of m/z396 underwent facile loss of water to give ions of m/z 513 and 378, consistent with aliphatic hydroxylation. The negative ion spectrum (data not shown) shows a deprotonated molecular ion ofm/z 529 that underwent pentanoic acid side chain loss to give an ion of m/z 429. These data suggest that oxidation has occurred on both the aromatic moiety as above and the isopropyl side chain. Mass spectra of the remaining radioactive components show molecular ions consistent with unsaturated analogues of the known metabolites, with molecular weights shifted lower by 2 mass units. Other radioactive peaks tentatively identified included unsaturated β-oxidized hydroxy AT of m/z 513 ([M + H]+, 86.4 min) and unsaturated β-oxidized AT of m/z 497 ([M + H]+, 87.1 min).

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Table 5

Atmospheric pressure chemical ionization mass spectra of mouse fecal metabolites

Figure 5
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Figure 5

ESI of a mouse feces component with an LC retention time of 68.5 min.

A, positive ion mass spectrum of m/z 573 [M+H]+. B, negative ion mass spectrum ofm/z 571 [M-H]−.

Figure 6
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Figure 6

APcI mass spectrum (cone voltage of 40V) of a component in mouse feces (LC retention time of 83.5 min) of m/z 531 [M+H]+.

Enzyme Induction Investigations.

Total spectral CYP450 concentrations increased by 5 and 30% in the 1.0 and 200 mg/kg treatment groups compared with control animals. Incubations with liver microsomes using ER, PR, testosterone, and lauric acid were employed as indicators of catalytic activity of CYP1A, -2B, -3A, and -4A, respectively. The results in table6 indicate that CYP1A showed a minor dose-dependent decrease in ER activity at the 200 mg/kg AT dose. The catalytic activity of CYP2B was variable with an initial increase in activity at the lower AT dose and a decrease at the higher one. CYP3A was the only CYP450 that demonstrated a small increase in activity with increasing AT dose, i.e. 26% increase at 200 mg/kg of AT. CYP4A activity, as measured by the disappearance of lauric acid, decreased to 83 and 47% at the 1 and 200 mg/kg AT doses, respectively.

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Table 6

Total spectral cytochrome P450 concentration and percent change in catalytic activities of CYP1A, -2B, -3A, and -4A from liver microsomes obtained from control mice and mice treated orally for 14 days at 1 or 200 mg/kg AT

Discussion

This study was performed to evaluate in vivo AT metabolism, total CYP450 levels, and selected CYP450 activities in mice after an earlier multiple-dose toxicokinetic study demonstrated marked dose-related effects on plasma HMG-CoA reductase inhibitory activity. Comparison of plasma radioequivalent concentrations after [14C]AT on days 1 and 14 showed a dramatic decline. Prior studies (Shum et al., 1996) have shown that the dosing duration altered neither half-life nor the time totmax. Recovery of radioactivity was essentially complete within 48 hr after either dosing regimen, with greater than 98% of the dose recovered in feces. In general, metabolite profiles of plasma and fecal extracts after both single- and multiple-dose administration were similar. Because of very low plasma concentrations, we only were able to examine the 1-hr plasma sample. After multiple doses of AT, two additional radioactive peaks were present that were not found after a single dose; however, whether these peaks represent new or additional metabolites could not be determined. Most metabolites present in plasma extracts were determined to be products of β-oxidation of the seven-carbon side chain. Minor peaks had also undergone aromatic hydroxylation. Identified in plasma extracts were AT (a minor component), a β-oxidized hydroxy metabolite of AT, β-oxidized AT, and unsaturated analogs of β-oxidized metabolites. All radioactive components identified in plasma extracts were found in fecal extracts. Additional fecal metabolites wereortho- and para-hydroxy AT and other unsaturated products including AT. Michniewicz et al. (1994) reported that ortho- and para-hydroxy AT were major metabolites in rat and dog bile; however, these metabolites were present in very small amounts.

In the present studies, we found only a 5 and 30% increase in total spectral CYP450 concentration at the 1.0 and 200 mg/kg AT levels, respectively. In addition, small and/or fluctuating changes were observed in the catalytic activities of CYP1A1, -2B, and -3A. Only CYP3A showed an increase in catalytic activity with an increasing AT dose. These minor increases in total CYP450 and CPY3A activity at the highest dose of AT seem insufficient to explain entirely the discrepancy observed in the plasma radioactivity concentrations between mice receiving single and multiple doses. In contrast, the CYP4A activity decreased dramatically to 47% of control at the 200 mg/kg AT dose level. More extensive experiments are needed to elucidate the mechanism of the decrease in CYP4A activity after multiple-dose AT administration.

Metabolism by the β-oxidation pathway in rodents has been observed with other HMG-CoA reductase inhibitors. β-Oxidation of the dihydroxy side chain (as the open ring form) plays a role in the metabolism of lovastatin, simvastatin, and pravastatin (Halpin et al., 1993, Komai et al., 1992, Vickers et al., 1990,Vyas et al., 1990). With the synthetically made fluvastatin, the extent of metabolism is greatest in the mouse; however, detailed examination of the metabolites was not provided (Tse et al., 1990). In the dog, β-oxidation of lovastatin, simvastatin, and pravastatin is a very minor metabolic pathway (Vickers et al., 1990). In addition, after oral administration of [14C]AT to bile fistula rats and dogs, examination of the bile indicates that β-oxidation is not a major pathway in these species (Michniewicz et al., 1994). The major metabolites identified in rat and dog resulted from aromatic hydroxylation to ortho- and para-hydroxy AT. In the mouse, although aromatic hydroxylation occurs, the major contribution is by β-oxidation of these and other metabolites. That unsaturated AT and other unsaturated metabolites contribute to the majority of radioactivity in fecal extracts does not seem to be an artifact. These unsaturated metabolites have not been observed in similar extracts from other species.

The decrease in plasma concentrations after multiple-dose administration cannot be explained entirely by induction of the CYP450 enzymes examined. The expected shorter plasma half-life of AT inhibitory equivalents and radioequivalents, a pharmacokinetic feature of induction, was not detected after multiple-dose administration in the mouse. Other HMG-CoA reductase inhibitors have not shown a decrease in plasma-equivalent concentrations after multiple-dose administration. CYP3A in the gut wall does not seem to contribute significantly to AT metabolism in the mouse because most of the metabolites detected result from β-oxidation rather than metabolism by CYP3A. The source of the effect seems more complicated than simple induction of hepatic cytochrome P450s; perhaps transport or β-oxidation is involved. The induction of β-oxidation, which can occur in mitochondria and peroxisomes (Osmundsen et al., 1991), was not examined in this study. Recently, other investigators have reported the effects of HMG-CoA reductase inhibitors on fatty acid metabolism in rodent liver. Studies in rats administered simvastatin indicate that this drug is not a peroxisomal proliferator; fatty acid distribution, however, was altered in microsomal phosphatidylcholines (Mercenne et al., 1991). Comparison of in vitro and in vivo effects of lovastatin on fatty acid metabolism in rat liver indicate an increased fatty acid-oxidative capacity (Guzman et al., 1993).

Metabolites detected after AT administration indicate that metabolism in the mouse is much more extensive (fig.7) with a greater predisposition toward β-oxidation than either rat or dog. AT disposition in mice is affected by multiple-dose treatment, something not explained by CYP450 levels or activity. Clearly, the β-oxidation pathway is favored in the mouse, but more detailed metabolism studies in mice are needed to assess the role of multiple-dose administration on this pathway.

Figure 7
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Figure 7

Atorvastatin and identified metabolites in mouse.

a indicates metabolites found in plasma;b indicates those found in feces.

Acknowledgments

The authors gratefully acknowledge the mass spectral work done by Dr. Rasmy Talaat of Covance Labs, Madison, WI, and the technical assistance of Mr. Robert Bonczyk of the Parke-Davis Pharmacokinetic/Drug Metabolism Department.

Footnotes

  • Send reprint requests to: Ann E. Black, Parke-Davis Pharmaceutical Research, 2800 Plymouth Rd., Ann Arbor, MI 48106.

  • Abbreviations used are::
    AT
    atorvastatin
    HMG
    3-hydroxy-3-methylglutaryl
    CID
    collision-induced dissociation
    APcI
    atmospheric pressure ionization
    ESI
    electrospray ionization
    ER
    ethoxyresorufin
    PR
    pentoxyresorufin
    CYP450
    cytochrome P450
    • Received August 4, 1997.
    • Accepted April 8, 1998.
  • The American Society for Pharmacology and Experimental Therapeutics

References

  1. ↵
    1. Baumann KL,
    2. Butler DE,
    3. Deering CF,
    4. Mennen KE,
    5. Millar A,
    6. Nanninga TN,
    7. Palmer CW,
    8. Roth BD
    (1992) The convergent synthesis of CI-981, an optically active, highly potent, tissue selective inhibitor of HMGCoA reductase. Tetrahedron Lett 33:2283–2284.
    OpenUrlCrossRef
  2. ↵
    Bjorge SM, Black AE, Roth DB and Woolf TF. U. S. Patent 5,385,929. Jan 31, 1995..
  3. ↵
    1. Black AE,
    2. Sinz MW,
    3. Hayes RN,
    4. Woolf TF
    (1995) In vivo and in vitro metabolism studies of [14C]CI-981 (AT) in male B6C3F1 mice. ISSX Proc 8:49.
  4. ↵
    1. Bradford MN
    (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254.
    OpenUrlCrossRefPubMed
  5. ↵
    1. Burke MD,
    2. Thompson S,
    3. Elcombe CR,
    4. Halpert J,
    5. Haaparanta T,
    6. Mayer RT
    (1985) Ethoxy-, pentoxy- and benzyloxyphenoxazones and homologues: A series of substrates to distinguish between different induced cytochromes P-450. Biochem Pharmacol 34:3337–3345.
    OpenUrlCrossRefPubMed
  6. ↵
    1. Hayes AW
    1. Guengerich FP
    (1989) Analysis and characterization of enzymes. in Principle and Methods of Toxicology, ed Hayes AW (Raven Press, New York), pp 777–814.
  7. ↵
    1. Guzman M,
    2. Cortes JP,
    3. Castro J
    (1993) Effects of lovastatin on hepatic fatty acid metabolism. Lipids 28:1087–1093.
    OpenUrlCrossRefPubMed
  8. ↵
    1. Halpin RA,
    2. Ulm EH,
    3. Till AE,
    4. Kari PH,
    5. Vyas KP,
    6. Hunninghake DB,
    7. Duggan DE
    (1993) Biotransformation of lovastatin: V. Species differences in vivo metabolite profiles of mouse, rat, dog, and human. Drug Metab Dispos 21:1003–1011.
    OpenUrlAbstract
  9. ↵
    1. Komai T,
    2. Kawai K,
    3. Tokui T,
    4. Tokui Y,
    5. Kuroiwa C,
    6. Shigehara E,
    7. Tanaka M
    (1992) Disposition and metabolism of pravastatin sodium in rats, dogs, and monkeys. Eur J Drug Metab Pharmacokinet 17:103–111.
    OpenUrlPubMed
  10. ↵
    1. Mercenne F,
    2. Goudonnet H,
    3. Mounie J,
    4. Escousse A,
    5. Truchot RC
    (1991) Effects of simvastatin, a lipoprotein-lowering drug, on the hepatic enzymes involved in drug metabolism in the Wistar rat. Xenobiotica 21:859–864.
    OpenUrlPubMed
  11. ↵
    1. Michniewicz BM,
    2. Black AE,
    3. Sinz MW,
    4. Woolf TF
    (1994) In vitro and in vivo metabolism of AT (CI-981). ISSX Proc 6:93.
    OpenUrl
  12. ↵
    1. Nawrocki JW,
    2. Weiss SR,
    3. Davidson MH,
    4. Sprecher DL,
    5. Schwartz SL,
    6. Lupien P,
    7. Jones PH,
    8. Haber HE,
    9. Black DM
    (1995) Reduction of LDL cholesterol by 25% to 60% in patients with primary hypercholesterolemia by Atorvastatin, a new HMG-CoA reductase inhibitor. Arterioscler Throm Vasc Biol 15:678–682.
    OpenUrlAbstract/FREE Full Text
  13. ↵
    1. Omura T,
    2. Sato R
    (1964) The carbon monoxide-binding pigment of liver microsomes. J Biol Chem 23:2379–2385.
    OpenUrl
  14. ↵
    1. Osmundsen H,
    2. Bremer J,
    3. Pedersen JI
    (1991) Metabolic aspects of peroxisomal beta-oxidation. Biochim Biophys Acta 1085:141–158.
    OpenUrlPubMed
  15. ↵
    Roth BD. U. S. Patent 4,681,893. July 21, 1987..
  16. ↵
    1. Roth BD,
    2. Blankley CJ,
    3. Chucholowski AW,
    4. Ferguson E,
    5. Hoefle ML,
    6. Ortwine DF,
    7. Newton RS,
    8. Sekerke CC,
    9. Sliskovic DR,
    10. Stratton CD,
    11. Wilson MW
    (1991) Inhibitors of cholesterol biosynthesis: 3. Tetrahydro-4-hydroxy-6-[2-(1H-pyrrol-1-yl)ethyl]2H-pyran-2-one inhibitors of HMC-CoA reductase. 2. Effects of introducing substituents at position three and four of the pyrrole nucleus. J Med Chem 34:357–366.
    OpenUrlCrossRefPubMed
  17. ↵
    1. Shum YY,
    2. Huang NJ,
    3. Whitfield LR
    (1996) Pharmacokinetics of atorvastatin in rodents. Pharm Res 13:S486.
    OpenUrl
  18. ↵
    1. Tse FL,
    2. Smith HT,
    3. Ballard FH,
    4. Nicoletti J
    (1990) Disposition of fluvastatin, an inhibitor of HMG-CoA reductase, in mouse, rat, dog, and monkey. Biopharm Drug Dispos 11:519–531.
    OpenUrlCrossRefPubMed
  19. ↵
    1. Vickers S,
    2. Duncan CA,
    3. Vyas KP,
    4. Kari PH,
    5. Arison B,
    6. Prakash SR,
    7. Ramjit HG,
    8. Pitzenberger SM,
    9. Stokker G,
    10. Duggan DE
    (1990) In vitro and in vivo biotransformation of simvastatin, an inhibitor of HMG CoA Reductase. Drug Metab Dispos 18:476–483.
    OpenUrlAbstract
  20. ↵
    1. Vyas KP,
    2. Kari PH,
    3. Pitzenberger SM,
    4. Halprin RA,
    5. Ramjit HG,
    6. Arison B,
    7. Murphy JS,
    8. Hoffman WR,
    9. Schwartz MS,
    10. Ulm EH,
    11. Duggan DE
    (1990) Biotransformation of Lovastatin: I. Structure elucidation of in vitro and in vivo metabolites in the rat and mouse. Drug Metab Dispos 18:203–211.
    OpenUrlAbstract
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Drug Metabolism and Disposition
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1 Aug 1998
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Research ArticleArticle

Metabolism and Excretion Studies in Mouse After Single and Multiple Oral Doses of the 3-Hydroxy-3-methylglutaryl-CoA Reductase Inhibitor Atorvastatin

Ann E. Black, Michael W. Sinz, Roger N. Hayes and Thomas F. Woolf
Drug Metabolism and Disposition August 1, 1998, 26 (8) 755-763;

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Research ArticleArticle

Metabolism and Excretion Studies in Mouse After Single and Multiple Oral Doses of the 3-Hydroxy-3-methylglutaryl-CoA Reductase Inhibitor Atorvastatin

Ann E. Black, Michael W. Sinz, Roger N. Hayes and Thomas F. Woolf
Drug Metabolism and Disposition August 1, 1998, 26 (8) 755-763;
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