Comparison of In Vitro and In Vivo Inhibition Potencies of Fluvoxamine toward CYP2C19

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This article is available online at http://dmd.aspetjournals.org ABSTRACT:The goal of the study was to test the assumption that a competitive inhibition constant determined in vivo, K i iv, like its corresponding in vitro counterpart, K i , is independent of inhibitor concentration. Inhibition of the CYP2C9-dependent formation of (S)-7-hydroxywarfarin from (S)-warfarin was measured in seven healthy subjects at three different doses of fluconazole. Prothrombin time measurements showed increasing anticoagulant activity with increasing fluconazole dose. The pharmacokinetic parameters calculated from the (S)-and (R)-warfarin plasma levels were consistent with previous studies. Fluconazole reduced the clearance of (S)-warfarin to a greater extent than that of (R)-warfarin. The decrease in clearance of both warfarin enantiomers was fluconazole dosedependent. The formation of (S)-7-hydroxywarfarin was inhibited by 31, 55, and 77% at the 100, 200, and 300 mg daily doses of fluconazole, respectively. K i iv, values calculated from these data based on plasma fluconazole levels at each dose and data from earlier work at 400-mg daily doses of fluconazole were 30.7 ؎ 23.7, 19.6 ؎ 3.8, 17.9 ؎ 7.5, and 19.8 ؎ 3.5 M, respectively. These results confirm the hypothesis that K i iv is independent of inhibitor concentration.In the last decade numerous studies have appeared that attempt to rationalize and correlate the metabolic behavior of drugs in vivo based on in vitro metabolic data. Encouraging results from initial studies prompted the Food and Drug Administration to publish two guidance for industry, one in 1997 [Department of Health and Human Services (1997)] and the second in 1999 [Department of Health and Human Services (1999)]. The guidance recommend that in vitro metabolic studies be conducted on promising new drugs early in the discovery process to determine their substrate and/or inhibitory properties toward the human P450s. 1The implicit assumption of the guidance and indeed of all in vitro -in vivo correlations is that the qualitative behaviors of the P450s are largely independent of their external biochemical environments. Thus, the same P450 should form the same primary metabolites from a given substrate and an inhibitor should inhibit the same enzymes, both in vitro and in vivo (Wrighton et al., 1993;Houston, 1994;Rodrigues, 1994;Ball et al., 1995;Houston and Carlile, 1997;Iwatsubo et al., 1997;Rodrigues and Wong, 1997;Ito et al., 1998). The assumption is essential because without it, in vitro -in vivo correlations would not be possible. Accumulated evidence suggests that in general it does hold, at least qualitatively. Quantitatively, the picture is much less clear and often problematic.Of the possible in vitro kinetic inhibition parameters that might be used to predict the magnitude of an in vivo drug interaction resulting from competitive inhibition, the in vitro K i is particularly useful. It is a constant that can be readily determined.2 Moreover, it is a constant that is largely independent of substrate ident...

Section: Formation Clearances To Each Of the Hydroxylated Metabolitessupporting

confidence: 72%

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confidence: 99%

K_IIV, AN IN VIVO PARAMETER FOR PREDICTING THE MAGNITUDE OF A DRUG INTERACTION ARISING FROM COMPETITIVE ENZYME INHIBITION

Neal¹,

Kunze²,

O’Reilly³

et al. 2003

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“…Inhibition of P450 enzymes is frequently the underlying mechanism for drug-drug interactions. Our understanding of this area has progressed to the extent that there exists confidence in the use of in vitro inhibition data for qualitative projection of drug-drug interactions in vivo; however, the science has not yet developed to the extent that quantitative projections of interactions can reliably be made from in vitro data (Yao and Levy, 2002;Yao et al, 2003). Nevertheless, in vitro inhibition data can be used in the planning of drug interaction studies in humans (Tucker et al, 2001).…”

Section: Discussionmentioning

confidence: 99%

Validated Assays for Human Cytochrome P450 Activities

Walsky

Obach²

2004

468

380

ABSTRACT:The measurement of the effect of new chemical entities on human cytochrome P450 marker activities using in vitro experimentation represents an important experimental approach in drug development. In vitro drug interaction data can be used in guiding the design of clinical drug interaction studies, or, when no effect is observed in vitro, the data can be used in place of an in vivo study to claim that no interaction will occur in vivo. To make such a claim, it must be assured that the in vitro experiments are performed with absolute confidence in the methods used and data obtained. To meet this need, 12 semiautomated assays for human P450 marker substrate activities have been developed and validated using approaches described in the GLP (good laboratory practices) as per the code of U.S. Federal Regulations. The assays that were validated are: phenacetin O-deethylase (CYP1A2), coumarin 7-hydroxylase (CYP2A6), bupropion hydroxylase (CYP2B6), amodiaquine N-deethylase (CYP2C8), diclofenac 4-hydroxylase and tolbutamide methylhydroxylase (CYP2C9), (S)-mephenytoin 4-hydroxylase (CYP2C19), dextromethorphan O-demethylase (CYP2D6), chlorzoxazone 6-hydroxylase (CYP2E1), felodipine dehydrogenase, testosterone 6␤-hydroxylase, and midazolam 1-hydroxylase (CYP3A4 and CYP3A5). High-pressure liquid chromatography-tandem mass spectrometry, using stable isotope-labeled internal standards, has been applied as the analytical method. This analytical approach, through its high sensitivity and selectivity, has permitted the use of very low incubation concentrations of microsomal protein (0.01-0.2 mg/ml). Analytical assay accuracy and precision values were excellent. Enzyme kinetic and inhibition parameters obtained using these methods demonstrated high precision and were within the range of values previously reported in the scientific literature. These methods should prove useful in the routine assessments of the potential for new drug candidates to elicit pharmacokinetic drug interactions via inhibition of cytochrome P450 activities.Drug-drug interactions are of great interest to scientists involved in drug research, regulatory authorities who are responsible for public safety, physicians, and their patients. Since "polypharmacy," or the practice of simultaneous prescription of more than one drug to treat one or more conditions in a single patient, has become a more common practice, drug interactions have been cited as one of the major reasons for hospitalization and even death (Lazarou et al., 1998). Thus, a great deal of effort is expended by researchers engaged in new drug research in avoiding the development of compounds that will cause drug-drug interactions.The most common mechanism underlying drug-drug interactions is the inhibition of cytochrome P450 activities. Several drugs in common use cause large increases in exposure to other drugs. Examples include ketoconazole, itraconazole, erythromycin, clarithromycin, diltiazem, and nefazodone (CYP3A); quinidine, paroxetine, and terbinafine (CYP2D6); ticlopidine (CYP2C19); ...

“…The predicted degree of interaction caused by a reversibly acting inhibitor can then be determined based on the ratio of the inhibitor concentration at the active site in vivo to the K i determined in vitro (Ito et al, 1998a,b). The majority of inhibition predictions in the literature have been performed using microsomal data (Yamano et al, 1999a(Yamano et al, ,b, 2000(Yamano et al, , 2001Komatsu et al, 2000;Ishigam et al, 2001;Tran et al, 2002;Yao et al, 2003 vivo clearance compared with microsomes (Houston, 1994), there is, at present, a lack of evidence to demonstrate that hepatocytes are better predictors of inhibition potential. There has been much controversy in the literature as to whether the total plasma, unbound plasma, or liver inhibitor concentration should be used for in vitro-in vivo extrapolations of inhibition (Bertz and Granneman, 1997;Ito et al, 1998a,b;Lin, 2000;Weaver, 2001).…”

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confidence: 99%

Quantitative Prediction of the in Vivo Inhibition of Diazepam Metabolism by Omeprazole Using Rat Liver Microsomes and Hepatocytes

Jones

Hallifax²,

Houston³

2004

ABSTRACT:The diazepam (DZ)-omeprazole (OMP) interaction has been selected as a prototype for an important drug-drug interaction involving cytochrome P450 inhibition. The availability of an in vivo K i value (unbound K i , 21 M) obtained from a series of steady-state inhibitor infusion studies allowed assessment of several in vitroderived predictions of this inhibition interaction. Studies monitoring substrate depletion with time were used to obtain in vitro K i values that were evaluated against the more traditional metabolite formation approach using microsomes and hepatocytes. OMP in- The inhibition of diazepam (DZ 1 ) metabolism by omeprazole (OMP) has been well documented in vivo; therapeutic concentrations of OMP have been reported to decrease the plasma clearance of DZ both in humans (Gugler and Jensen, 1985; Andersson et al., 1990a,b) and in rats . measured the in vivo clearance of DZ (administered by the hepatic portal vein) in rats receiving intravenous infusions together with matching bolus doses of OMP to achieve a wide range of steady-state plasma concentrations (10 -50 g/ml). The inhibition was modeled using a simple inhibition model (eq. 1) and the in vivo K i was calculated to be 57 M.where CL and CL 0 represent DZ clearance in the presence and absence of OMP, respectively, I ss represents the steady-state total plasma concentration of OMP, and K i represents the inhibition constant. Figure 1 shows the relationship between DZ clearance and steady-state unbound concentration of OMP achieved in this study. Such a detailed in vivo inhibition study is only possible in animals. In humans, inhibition studies are normally performed using one inhibitor oral dosing regimen, and the ratio of the AUC in the presence and absence of the inhibitor is used to assess the degree of interaction (Gugler and Jensen, 1985;