Interaction of cisapride with the human cytochrome P450 system

Metabolism and inhibition studies

Zeruesenay Desta, Nadia Soukhova, Subena K. Mahal, David A. Flockhart

Research output: Contribution to journalArticle

43 Citations (Scopus)

Abstract

Using human liver microsomes (HLMs) and recombinant cytochrome P450s (CYP450s), we characterized the CYP450 isoforms involved in the primary metabolic pathways of cisapride and documented the ability of cisapride to inhibit the CYP450 system. In HLMs, cisapride was N-dealkylated to norcisapride (NORCIS) and hydroxylated to 3-fluoro-4-hydroxycisapride (3-F-4- OHCIS) and to 4-fluoro-2-hydroxycisapride (4-F-2-OHCIS). Formation of NORCIS, 3-F-4-OHCIS, and 4-F-2-OHCIS in HLMs exhibited Michaelis-Menten kinetics (K(m): 23.4 ± 8.6, 32 ± 11, and 31 ± 23 μM; V(max): 155 ± 91, 52 ± 23, and 31 ± 23 pmol/min/mg of protein, respectively). The average in vitro intrinsic clearance (V(max)/K(m)) revealed that the formation of NORCIS was 3.9- to 5.9-fold higher than that of the two hydroxylated metabolites. Formation rate of NORCIS from 10 μM cisapride in 14 HLMs was highly variable (range, 4.9-133.6 pmol/min/mg of protein) and significantly correlated with the activities of CYP3A (r = 0.86, P = .0001), CYP2C19, and 1A2. Of isoform- specific inhibitors, 1 μM ketoconazole and 50 μM troleandomycin were potent inhibitors of NORCIS formation from 10 μM cisapride (by 51 ± 9 and 44 ± 17%, respectively), whereas the effect of other inhibitors was minimal. Of 10 recombinant human CYP450s tested, CYP3A4 formed NORCIS from 10 μM cisapride at the highest rate (V = 0.56 ± 0.13 pmol/min/pmol of P450) followed by CYP2C8 (V = 0.29 - 0.08 pmol/min/pmol of P450) and CYP2B6 (0.15 ± 0.04 pmol/min/pmol of P450). The formation of 3-F-4-OHCIS was mainly catalyzed by CYP2C8 (V = 0.71 ± 0.24 pmol/min/pmol of P450) and that of 4-F-2-OHCIS by CYP3A4 (0.16 ± 0.03 pmol/min/pmol of P450). Clearly, recombinant CYP2C8 participates in cisapride metabolism, but when the in vitro intrinsic clearances obtained were corrected for abundance of each CYP450 in the liver, CYP3A4 is the dominant isoform. Cisapride was a relatively potent inhibitor of CYP2D6, with no significant effect on other isoforms tested, but the K(i) value derived (14 ± 16 μM) was much higher than the clinically expected concentration of cisapride (

Original languageEnglish (US)
Pages (from-to)789-800
Number of pages12
JournalDrug Metabolism and Disposition
Volume28
Issue number7
StatePublished - 2000
Externally publishedYes

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Cisapride
Enzyme inhibition
Metabolism
Cytochrome P-450 Enzyme System
Cytochrome P-450 CYP3A
Liver
Liver Microsomes
Protein Isoforms
Cytochromes
Troleandomycin
Ketoconazole
Metabolites
Metabolic Networks and Pathways
norcisapride
Proteins

ASJC Scopus subject areas

  • Pharmacology
  • Toxicology

Cite this

Interaction of cisapride with the human cytochrome P450 system : Metabolism and inhibition studies. / Desta, Zeruesenay; Soukhova, Nadia; Mahal, Subena K.; Flockhart, David A.

In: Drug Metabolism and Disposition, Vol. 28, No. 7, 2000, p. 789-800.

Research output: Contribution to journalArticle

Desta, Zeruesenay ; Soukhova, Nadia ; Mahal, Subena K. ; Flockhart, David A. / Interaction of cisapride with the human cytochrome P450 system : Metabolism and inhibition studies. In: Drug Metabolism and Disposition. 2000 ; Vol. 28, No. 7. pp. 789-800.
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title = "Interaction of cisapride with the human cytochrome P450 system: Metabolism and inhibition studies",
abstract = "Using human liver microsomes (HLMs) and recombinant cytochrome P450s (CYP450s), we characterized the CYP450 isoforms involved in the primary metabolic pathways of cisapride and documented the ability of cisapride to inhibit the CYP450 system. In HLMs, cisapride was N-dealkylated to norcisapride (NORCIS) and hydroxylated to 3-fluoro-4-hydroxycisapride (3-F-4- OHCIS) and to 4-fluoro-2-hydroxycisapride (4-F-2-OHCIS). Formation of NORCIS, 3-F-4-OHCIS, and 4-F-2-OHCIS in HLMs exhibited Michaelis-Menten kinetics (K(m): 23.4 ± 8.6, 32 ± 11, and 31 ± 23 μM; V(max): 155 ± 91, 52 ± 23, and 31 ± 23 pmol/min/mg of protein, respectively). The average in vitro intrinsic clearance (V(max)/K(m)) revealed that the formation of NORCIS was 3.9- to 5.9-fold higher than that of the two hydroxylated metabolites. Formation rate of NORCIS from 10 μM cisapride in 14 HLMs was highly variable (range, 4.9-133.6 pmol/min/mg of protein) and significantly correlated with the activities of CYP3A (r = 0.86, P = .0001), CYP2C19, and 1A2. Of isoform- specific inhibitors, 1 μM ketoconazole and 50 μM troleandomycin were potent inhibitors of NORCIS formation from 10 μM cisapride (by 51 ± 9 and 44 ± 17{\%}, respectively), whereas the effect of other inhibitors was minimal. Of 10 recombinant human CYP450s tested, CYP3A4 formed NORCIS from 10 μM cisapride at the highest rate (V = 0.56 ± 0.13 pmol/min/pmol of P450) followed by CYP2C8 (V = 0.29 - 0.08 pmol/min/pmol of P450) and CYP2B6 (0.15 ± 0.04 pmol/min/pmol of P450). The formation of 3-F-4-OHCIS was mainly catalyzed by CYP2C8 (V = 0.71 ± 0.24 pmol/min/pmol of P450) and that of 4-F-2-OHCIS by CYP3A4 (0.16 ± 0.03 pmol/min/pmol of P450). Clearly, recombinant CYP2C8 participates in cisapride metabolism, but when the in vitro intrinsic clearances obtained were corrected for abundance of each CYP450 in the liver, CYP3A4 is the dominant isoform. Cisapride was a relatively potent inhibitor of CYP2D6, with no significant effect on other isoforms tested, but the K(i) value derived (14 ± 16 μM) was much higher than the clinically expected concentration of cisapride (",
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T1 - Interaction of cisapride with the human cytochrome P450 system

T2 - Metabolism and inhibition studies

AU - Desta, Zeruesenay

AU - Soukhova, Nadia

AU - Mahal, Subena K.

AU - Flockhart, David A.

PY - 2000

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N2 - Using human liver microsomes (HLMs) and recombinant cytochrome P450s (CYP450s), we characterized the CYP450 isoforms involved in the primary metabolic pathways of cisapride and documented the ability of cisapride to inhibit the CYP450 system. In HLMs, cisapride was N-dealkylated to norcisapride (NORCIS) and hydroxylated to 3-fluoro-4-hydroxycisapride (3-F-4- OHCIS) and to 4-fluoro-2-hydroxycisapride (4-F-2-OHCIS). Formation of NORCIS, 3-F-4-OHCIS, and 4-F-2-OHCIS in HLMs exhibited Michaelis-Menten kinetics (K(m): 23.4 ± 8.6, 32 ± 11, and 31 ± 23 μM; V(max): 155 ± 91, 52 ± 23, and 31 ± 23 pmol/min/mg of protein, respectively). The average in vitro intrinsic clearance (V(max)/K(m)) revealed that the formation of NORCIS was 3.9- to 5.9-fold higher than that of the two hydroxylated metabolites. Formation rate of NORCIS from 10 μM cisapride in 14 HLMs was highly variable (range, 4.9-133.6 pmol/min/mg of protein) and significantly correlated with the activities of CYP3A (r = 0.86, P = .0001), CYP2C19, and 1A2. Of isoform- specific inhibitors, 1 μM ketoconazole and 50 μM troleandomycin were potent inhibitors of NORCIS formation from 10 μM cisapride (by 51 ± 9 and 44 ± 17%, respectively), whereas the effect of other inhibitors was minimal. Of 10 recombinant human CYP450s tested, CYP3A4 formed NORCIS from 10 μM cisapride at the highest rate (V = 0.56 ± 0.13 pmol/min/pmol of P450) followed by CYP2C8 (V = 0.29 - 0.08 pmol/min/pmol of P450) and CYP2B6 (0.15 ± 0.04 pmol/min/pmol of P450). The formation of 3-F-4-OHCIS was mainly catalyzed by CYP2C8 (V = 0.71 ± 0.24 pmol/min/pmol of P450) and that of 4-F-2-OHCIS by CYP3A4 (0.16 ± 0.03 pmol/min/pmol of P450). Clearly, recombinant CYP2C8 participates in cisapride metabolism, but when the in vitro intrinsic clearances obtained were corrected for abundance of each CYP450 in the liver, CYP3A4 is the dominant isoform. Cisapride was a relatively potent inhibitor of CYP2D6, with no significant effect on other isoforms tested, but the K(i) value derived (14 ± 16 μM) was much higher than the clinically expected concentration of cisapride (

AB - Using human liver microsomes (HLMs) and recombinant cytochrome P450s (CYP450s), we characterized the CYP450 isoforms involved in the primary metabolic pathways of cisapride and documented the ability of cisapride to inhibit the CYP450 system. In HLMs, cisapride was N-dealkylated to norcisapride (NORCIS) and hydroxylated to 3-fluoro-4-hydroxycisapride (3-F-4- OHCIS) and to 4-fluoro-2-hydroxycisapride (4-F-2-OHCIS). Formation of NORCIS, 3-F-4-OHCIS, and 4-F-2-OHCIS in HLMs exhibited Michaelis-Menten kinetics (K(m): 23.4 ± 8.6, 32 ± 11, and 31 ± 23 μM; V(max): 155 ± 91, 52 ± 23, and 31 ± 23 pmol/min/mg of protein, respectively). The average in vitro intrinsic clearance (V(max)/K(m)) revealed that the formation of NORCIS was 3.9- to 5.9-fold higher than that of the two hydroxylated metabolites. Formation rate of NORCIS from 10 μM cisapride in 14 HLMs was highly variable (range, 4.9-133.6 pmol/min/mg of protein) and significantly correlated with the activities of CYP3A (r = 0.86, P = .0001), CYP2C19, and 1A2. Of isoform- specific inhibitors, 1 μM ketoconazole and 50 μM troleandomycin were potent inhibitors of NORCIS formation from 10 μM cisapride (by 51 ± 9 and 44 ± 17%, respectively), whereas the effect of other inhibitors was minimal. Of 10 recombinant human CYP450s tested, CYP3A4 formed NORCIS from 10 μM cisapride at the highest rate (V = 0.56 ± 0.13 pmol/min/pmol of P450) followed by CYP2C8 (V = 0.29 - 0.08 pmol/min/pmol of P450) and CYP2B6 (0.15 ± 0.04 pmol/min/pmol of P450). The formation of 3-F-4-OHCIS was mainly catalyzed by CYP2C8 (V = 0.71 ± 0.24 pmol/min/pmol of P450) and that of 4-F-2-OHCIS by CYP3A4 (0.16 ± 0.03 pmol/min/pmol of P450). Clearly, recombinant CYP2C8 participates in cisapride metabolism, but when the in vitro intrinsic clearances obtained were corrected for abundance of each CYP450 in the liver, CYP3A4 is the dominant isoform. Cisapride was a relatively potent inhibitor of CYP2D6, with no significant effect on other isoforms tested, but the K(i) value derived (14 ± 16 μM) was much higher than the clinically expected concentration of cisapride (

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