Use of inhibitory monoclonal antibodies to assess the contribution of cytochromes P450 to human drug metabolism

Shou, Magang; Lu, Ting; Krausz, Kristopher W.; Sai, Yang; Yang, Tianjian; Korzekwa, Kenneth R.; Gonzalez, Frank J.; Gelboin, Harry V.

doi:10.1016/s0014-2999(00)00079-0

Cited by 43 publications

(37 citation statements)

References 25 publications

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“…Ascites fluid from mice producing inhibitory monoclonal antibodies against human CYP enzymes were obtained from the National Institutes of Health/National Cancer Institute (NIH/NCI)10,11. Control mouse ascites fluid (lyophilized) was purchased from Cedarlane Laboratories (Hornby, Ontario, Canada).…”

Section: Methodsmentioning

confidence: 99%

Identification of metabolites of dalfampridine (4-aminopyridine) in human subjects and reaction phenotyping of relevant cytochrome P450 pathways

Caggiano

Blight

2013

Journal of Drug Assessment

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ObjectivesAn extended release formulation of dalfampridine (4-aminopyridine; 4-AP), a potassium channel blocker is available in the USA to improve walking in patients with multiple sclerosis. This study investigated the human metabolites of 4-AP and the cytochrome P450 (CYP450) pathways responsible for 4-AP metabolism.MethodsMetabolites were identified, using thin layer chromatography, high performance liquid chromatography, and gas chromatography/mass spectroscopy, in plasma and urine samples obtained during an excretion balance study of four subjects who were administered a single oral 15-mg dose of 14C-4-AP. Samples were compared with authentic standards of 4-AP, 2-hydroxy-4AP, 3-hydroxy-4AP, and 4-AP-N-oxide. Reaction phenotyping was performed in vitro using human liver microsomes and recombinant CYP450 enzymes with incubation in the presence of direct and time-dependent inhibitors to determine the CYP450 pathways involved in metabolite formation.ResultsWhile most (∼70%) of the radioactivity detected in plasma at each time point corresponded to unchanged 4-AP, two major metabolites were recovered. One metabolite co-localized with the authentic reference standard of 3-hydroxy-4-AP, and the other metabolite was identified as the sulfate conjugate of 3-hydroxy-4-AP. Two minor components were observed, one accounting for 2% of radioactivity and the other below the level of quantitation. Reaction phenotyping showed moderate correlations for conversion of 4-AP to 3-hydroxy-4AP with both CYP2E1 (r = 0.596; p < 0.001) and CYP2C8 (r = 0.608; p < 0.001). Use of a CYP2E1 metabolism-dependent inhibitor inhibited formation of 3-hydroxy-4-AP with and without pre-incubation (higher inhibition with pre-incubation), further supporting the likelihood of CYP2E1 as a metabolic pathway. The main limitation of this study was the inability to identify the CYP enzymes responsible for the 3-hydroxylation of 4-AP, although this conversion represents only a minor metabolic pathway.ConclusionThere is limited metabolism of 4-AP in humans. The two major metabolites were 3-hydroxy-4-AP and 3-hydroxy-4-AP sulfate, likely through CYP2E1 pathways; the possibility of other CYP enzymes playing a minor role in 4-AP metabolism could not be established unequivocally. Overall, these data suggest that there is a low risk for drug–drug interactions via an impact on 4-AP metabolism through cytochrome pathways.

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Section: Methodsmentioning

confidence: 99%

Identification of metabolites of dalfampridine (4-aminopyridine) in human subjects and reaction phenotyping of relevant cytochrome P450 pathways

Caggiano

Blight

2013

Journal of Drug Assessment

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“…On the other hand, CYP3A4 is known as one of the major CYP enzymes in the liver, 13,[17][18][19][20] and in vitro studies using primate and human liver microsomes have demonstrated that AFQ 1 is a major AFB 1 metabolite.…”

Section: 3mentioning

confidence: 99%

“…2,[14][15][16] However, quantitative data on AFM 1 excretion into feces are limited. On the other hand, CYP3A4 is known as one of the major CYP enzymes in the liver, 13,[17][18][19][20] and in vitro studies using primate and human liver microsomes have demonstrated that AFQ 1 is a major AFB 1 metabolite. 21,22 Excretion of AFQ 1 , especially fecal excretion in humans, has not been quantitatively or qualitatively characterized.…”

Section: Guanine; Hepatitis B Virusmentioning

confidence: 99%

Fecal and urinary excretion of aflatoxin B₁ metabolites (AFQ₁, AFM₁ and AFB‐N⁷‐guanine) in young Chinese males

Mykkänen

Zhu

Salminen

et al. 2005

Intl Journal of Cancer

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Our study was designed to assess the fecal and urinary excretion of 3 aflatoxin B 1 (AFB 1 ) metabolites, aflatoxins M 1 (AFM 1 ) and Q 1 (AFQ 1 ) and aflatoxin B 1 -N 7 -guanine (AFB-N 7 -guanine) that are produced by the predominant forms of cytochrome P450 enzymes responsible for the biotransformation of AFB 1 . Fecal and urinary AFM 1 , AFQ 1 and urinary AFB-N 7 -guanine were assessed in 83 young Chinese males selected from a larger population (n = 300) based on detectable urinary AFM 1 . The concentration of fecal AFQ 1 (median 137 ng/g fresh weight, IQR 9.1 to 450) was approximately 60 times higher than that of AFM 1 (2.3 ng/g, IQR 0.0 to 7.3). In urine, the median AFQ 1 was 10.4 ng/ml (IQR 3.4 to 23.3), and the median AFM 1 and AFB-N 7 -guanine 0.04 ng/ml (IQR 0.01 to 0.33) and 0.38 ng/ml (IQR 0.0 to 2.15), respectively. A subgroup (n = 14) with hepatitis B virus (HBV) infection had significantly higher fecal concentrations of AFQ 1 ( p = 0.043) and AFM 1 ( p = 0.001) than those who were hepatitis B-virus antigen (HBsAg) negative, and the respective differences in urinary AFQ 1 and AFM 1 concentrations approached statistical significance ( p = 0.054, p = 0.138). Our study demonstrates that AFQ 1 is excreted in urine and feces at higher levels than AFM 1 , and feces are an important route of excretion of these AFB 1 metabolites. AFQ 1 should be further assessed for its predictive value as a marker for exposure and risk of dietary aflatoxins. The most serious health effect of dietary exposure to aflatoxins is hepatocellular carcinoma. The etiology of this disease also involves chronic infection with hepatitis B and C viruses (HBV and HCV), and a significant synergistic interaction between aflatoxin exposure and hepatitis B virus has been reported. 2,3After oral uptake AFB 1 is efficiently absorbed and metabolized prior to excretion by fecal and urinary routes (Fig. 1). Absorbed AFB 1 and its metabolites are excreted in urine, while elimination to feces is a route for both the unabsorbed AFB 1 and biliary excretion of metabolites formed from the absorbed toxin. Animal studies have shown that under normal conditions 50% of the orally administered dose of AFB 1 is quickly absorbed from the duodenal region of the small intestine 4 and enters the liver through the hepatic portal blood supply.5 AFB 1 is concentrated in the liver and to a lesser extent in kidney, 6 and it is also found in the mesenteric venous blood as free AFB 1 or its water-soluble metabolites.Members of the cytochrome P450 (CYP) enzyme family, CYP1A2, CYP3A4 and CYP2A6 have been shown to be responsible for the metabolism of the absorbed aflatoxins 7,8 (Fig. 1). These enzymes convert AFB 1 to its carcinogenic form, AFB-8,9-epoxide, which is covalently bound to DNA 9 and serum albumin, 10 forming aflatoxin B 1 -N 7 -guanine (AFB-N 7 -guanine) and lysine adducts, respectively. These enzymes also oxidize AFB 1 to various other derivatives, including aflatoxin M 1 (AFM 1 ), aflatoxin Q 1 (AFQ 1 ), aflatoxin P 1 as well as a reduced aflatoxin species...

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“…has been published by Gelboin's laboratory (Gelboin, 1993;Gelboin et al, 1995Gelboin et al, , 1996Gelboin et al, , 1997Gelboin et al, , 1998Gelboin et al, , 1999Yang et al, 1998a,b;Sai et al, 1999;Krausz et al, 2001). These antibodies are specific to the P450s with immunoblot and inhibitory properties, which can be used to assess expression level of the individual P450s in tissues in response to the treatment of an inducer and to quantitatively measure contribution of the P450s to the metabolism of a drug or chemical including toxins, mutagens, and carcinogens (Gelboin, 1993;Gelboin et al, 1998Gelboin et al, , 1999Yang et al, 1998cYang et al, , 1999Mei et al, 1999;Shou et al, 2000). Although MAbs have some advantages in P450 identification and quantification studies, there are a number of concerns in their application of MAbs.…”

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confidence: 99%

Antibodies as a Probe in Cytochrome P450 Research

Shou

Lu²

2009

Drug Metab Dispos

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ABSTRACT:Cytochrome P450 (P450) is the superfamily of enzymes responsible for biotransformation of endobiotics and xenobiotics. However, their large isoform multiplicity, inducibility, diverse structure, widespread distribution, polymorphic expression, and broad overlapping substrate specificity make it difficult to measure the precise role of each individual P450 to the metabolism of drugs (or carcinogens) and hamper the understanding of the relationship between the genetic/environmental factors that regulate P450 phenotype and the responses of the individual P450s to drugs. The antibodies against P450s have been useful tools for the quantitative determination of expression level and contribution of the epitope-specific P450 to the metabolism of a drug or carcinogen substrate in tissues containing multiple P450 isoforms and for implications in pharmacogenetics and human risk assessment. In particular, the inhibitory antibodies are uniquely suited for reaction phenotyping that helps to predict human pharmacokinetics for clinical drugdrug interaction potential in drug discovery and development.Many enzyme systems exist in the liver and in extrahepatic tissues, such as intestine and kidney, that can contribute to the clearance of drugs from the systemic circulation. Among the various phase I and phase II drug-metabolizing enzymes, the cytochrome P450 (P450) enzymes play a key role in xenobiotic and endobiotic metabolic processing. It has been estimated that approximately two thirds of marketed drugs are metabolized by this enzyme system (Rendic and Di Carlo, 1997;Guengerich, 2003;Williams et al., 2004). In fact, members of three P450 subfamilies (CYP1, CYP2, and CYP3) are largely responsible for the metabolic clearance of drugs and xenobiotics (Rendic and Di Carlo, 1997;Guengerich, 2003;Williams et al., 2004). Multiplicity and overlapping substrate specificity of P450 have greatly complicated efforts at understanding the precise role of individual P450 isoforms in the metabolism of drugs and drug candidates. Therefore, over recent years, a variety of reagents and tools have been developed for in vitro and in vivo studies, and it is now possible to determine which specific P450 isoform(s) is (are) involved in the metabolism of a given compound (Rodrigues, 1999;Rodrigues and Rushmore, 2002;Lu et al., 2003;Williams et al., 2003). Given the importance of fraction of drug metabolized (f m ) in assessing the potential of in vivo drug-drug interaction (DDI), P450 reaction-phenotyping studies are conducted at multiple stages during drug discovery and development. Irrespective of the strategy, information related to P450-mediated metabolism is included in regulatory documents and the absorption, distribution, metabolism, and elimination (ADME) and DDI sections of the product label, where precautions concerning the coadministration of other drugs are listed along with recommendations for dose adjustment (Obach et al., 2006;Huang et al., 2007). Antibodies that are specific to individual P450s can be used to determine P450 pr...

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Use of inhibitory monoclonal antibodies to assess the contribution of cytochromes P450 to human drug metabolism

Cited by 43 publications

References 25 publications

Identification of metabolites of dalfampridine (4-aminopyridine) in human subjects and reaction phenotyping of relevant cytochrome P450 pathways

Identification of metabolites of dalfampridine (4-aminopyridine) in human subjects and reaction phenotyping of relevant cytochrome P450 pathways

Fecal and urinary excretion of aflatoxin B₁ metabolites (AFQ₁, AFM₁ and AFB‐N⁷‐guanine) in young Chinese males

Antibodies as a Probe in Cytochrome P450 Research

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Use of inhibitory monoclonal antibodies to assess the contribution of cytochromes P450 to human drug metabolism

Cited by 43 publications

References 25 publications

Identification of metabolites of dalfampridine (4-aminopyridine) in human subjects and reaction phenotyping of relevant cytochrome P450 pathways

Identification of metabolites of dalfampridine (4-aminopyridine) in human subjects and reaction phenotyping of relevant cytochrome P450 pathways

Fecal and urinary excretion of aflatoxin B1 metabolites (AFQ1, AFM1 and AFB‐N7‐guanine) in young Chinese males

Antibodies as a Probe in Cytochrome P450 Research

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Fecal and urinary excretion of aflatoxin B₁ metabolites (AFQ₁, AFM₁ and AFB‐N⁷‐guanine) in young Chinese males