Use of in vitro human metabolism studies in drug development

Ad, Rodrigues

doi:10.1016/0006-2952(94)00312-2

Cited by 129 publications

(52 citation statements)

References 64 publications

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“…The conclusion of CYP3A4-selective catalysis is derived from the results of an integrated in vitro approach (30,31) based on: (a) analysis of the possible correlation between the rates of PNU-159682 formation and several CYP form-selective activities in a panel of HLMs from 10 subjects, (b) evaluation of the effects of CYP formselective chemical inhibitors and MAbs specific and inhibitory to different human hepatic CYPs, on MMDX conversion to PNU-159682 by pooled HLMs, and (c) evaluation of the intrinsic ability of individual recombinant human CYP enzymes to catalyze formation of the metabolite. Correlation studies showed that the rate of PNU-159682 formation was highly significantly correlated with three different CYP3A-mediated activities (Fig.…”

Section: Discussionmentioning

confidence: 99%

Formation and Antitumor Activity of PNU-159682, A Major Metabolite of Nemorubicin in Human Liver Microsomes

Quintieri

Geroni

Fantin

et al. 2005

Clinical Cancer Research

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Purpose: Nemorubicin (3V-deamino-3V-[2 00 (S)-methoxy-4 00 -morpholinyl]doxorubicin; MMDX) is an investigational drug currently in phase II/III clinical testing in hepatocellular carcinoma. A bioactivation product of MMDX, 3V-deamino-3 00 ,4V-anhydro-[2 00 (S)-methoxy-3 00 (R)-oxy-4 00 -morpholinyl]doxorubicin (PNU-159682), has been recently identified in an incubate of the drug with NADPHsupplemented rat liver microsomes. The aims of this study were to obtain information about MMDX biotransformation to PNU-159682 in humans, and to explore the antitumor activity of PNU-159682.Experimental Design: Human liver microsomes (HLM) and microsomes from genetically engineered cell lines expressing individual human cytochrome P450s (CYP) were used to study MMDX biotransformation. We also examined the cytotoxicity and antitumor activity of PNU-159682 using a panel of in vitro-cultured human tumor cell lines and tumor-bearing mice, respectively.Results: HLMs converted MMDX to a major metabolite, whose retention time in liquid chromatography and ion fragmentation in tandem mass spectrometry were identical to those of synthetic PNU-159682. In a bank of HLMs from 10 donors, rates of PNU-159682 formation correlated significantly with three distinct CYP3A-mediated activities. Troleandomycin and ketoconazole, both inhibitors of CYP3A, markedly reduced PNU-159682 formation by HLMs; the reaction was also concentration-dependently inhibited by a monoclonal antibody to CYP3A4/5. Of the 10 cDNA-expressed CYPs examined, only CYP3A4 formed PNU-159682. In addition, PNU-159682 was remarkably more cytotoxic than MMDX and doxorubicin in vitro, and was effective in the two in vivo tumor models tested, i.e., disseminated murine L1210 leukemia and MX-1 human mammary carcinoma xenografts.Conclusions: CYP3A4, the major CYP in human liver, converts MMDX to a more cytotoxic metabolite, PNU-159682, which retains antitumor activity in vivo.

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Section: Discussionmentioning

confidence: 99%

Formation and Antitumor Activity of PNU-159682, A Major Metabolite of Nemorubicin in Human Liver Microsomes

Quintieri

Geroni

Fantin

et al. 2005

Clinical Cancer Research

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“…1 The 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.…”

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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

Drug Metab Dispos

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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...

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“…Screening strategies early in lead optimization now typically incorporate highthroughput in vitro absorption, distribution, metabolism, and excretion (ADME 1 ) measurements alongside potency determinations. This early screening has occurred due to the increased compound synthesis from combinatorial chemistry, the increase in knowledge and availability of techniques for studying a compound's ADME properties, and, most importantly, the degree of attrition from unacceptable pharmacokinetics of drug candidates in the latter, more expensive stages of drug development (Rodrigues, 1994;Eddershaw and Dickins, 1999;Eddershaw et al, 2000;Thompson, 2001). While the incorporation of ADME assays into a screening strategy allows the generation of large amounts of data on many compounds, a rational drug metabolism strategy that arises from a sound understanding of the relevant in vitro techniques is preferable over a "brute force" approach of data generation.…”

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confidence: 99%

Flavin-Containing Monooxygenase Activity in Hepatocytes and Microsomes: In Vitro Characterization and In Vivo Scaling of Benzydamine Clearance

Fisher¹,

Yoon²,

Vaughn³

et al. 2002

Drug Metabolism and Disposition

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ABSTRACT:Liver microsomes, and more recently cryopreserved hepatocytes, are commonly used in the in vitro characterization of the metabolism of new xenobiotics. The flavin-containing monooxygenases (FMO) are a major nonP450 oxidase present in liver microsomes and hepatocytes. Since FMO is known to be thermally labile, and this enzyme may be involved in the metabolic clearance of some drugs, we sought to more completely characterize the metabolic competency of this enzyme in cryopreserved hepatocytes and in liver microsomes preincubated under various conditions using benzydamine as an in vitro and in vivo probe. The metabolism of benzydamine to its major metabolite, the N-oxide, is mediated by FMO3 in humans. We found that the in vitro microsomal t 1/2 was 70% longer when incubations were prewarmed at 37°C in the absence of NADPH compared with prewarming in the presence of an NADPH-regenerating system, and N-oxide formation was inhibited >99%. Interestingly, the in vivo clearance predicted from these incubations and from human hepatocytes overpredicted the observed clearance of benzydamine in humans (>10.5 versus 2.4 ml/min/kg). In contrast, rat hepatocytes successfully predicted rat in vivo benzydamine clearance to within ϳ30% (>68 versus 48 ml/min/kg). Benzydamine N-oxidation in liver microsomes from all common preclinical species demonstrated heat sensitivity. This information should be considered when extrapolating metabolism data of xenobiotics from these in vitro systems.

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Use of in vitro human metabolism studies in drug development

Cited by 129 publications

References 64 publications

Formation and Antitumor Activity of PNU-159682, A Major Metabolite of Nemorubicin in Human Liver Microsomes

Formation and Antitumor Activity of PNU-159682, A Major Metabolite of Nemorubicin in Human Liver Microsomes

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

Flavin-Containing Monooxygenase Activity in Hepatocytes and Microsomes: In Vitro Characterization and In Vivo Scaling of Benzydamine Clearance

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Use of in vitro human metabolism studies in drug development

Cited by 129 publications

References 64 publications

Formation and Antitumor Activity of PNU-159682, A Major Metabolite of Nemorubicin in Human Liver Microsomes

Formation and Antitumor Activity of PNU-159682, A Major Metabolite of Nemorubicin in Human Liver Microsomes

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

Flavin-Containing Monooxygenase Activity in Hepatocytes and Microsomes: In Vitro Characterization and In Vivo Scaling of Benzydamine Clearance

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