Mass Spectrometry-Based Quantification of CYP Enzymes to Establish In Vitro/In Vivo Scaling Factors for Intestinal and Hepatic Metabolism in Beagle Dog

Heikkinen, Aki T.; Friedlein, Arno; Lamerz, Jens; Jakob, Peter; Cutler, Paul; Fowler, Stephen; Williamson, Tara; Tolando, Roberto; Lavé, Thierry; Parrott, Neil

doi:10.1007/s11095-012-0707-7

Cited by 29 publications

(36 citation statements)

References 32 publications

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“…Figure 1 shows the distribution of the different CYPs in liver and small intestinal mucosa of human 9, 10 and dog 11 . The liver has the highest content of drug metabolizing CYPs and is the most important organ for CYP mediated drug elimination.…”

Section: Dog-human Cyp Similarities and Differencesmentioning

confidence: 99%

Canine Cytochrome P-450 Pharmacogenetics

Court

2013

Veterinary Clinics of North America: Small Animal Practice

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Synopsis The cytochrome P450 (CYP) drug metabolizing enzymes are essential for the efficient elimination of many clinically used drugs. These enzymes typically display high interindividual variability in expression and function resulting from enzyme induction, inhibition, and genetic polymorphism thereby predisposing patients to adverse drug reactions or therapeutic failure. There are also substantial species differences in CYP substrate specificity and expression that complicate direct extrapolation of information from humans to veterinary species. This article reviews the available published data regarding the presence and impact of genetic polymorphisms on CYP-dependent drug metabolism in dogs in the context of known human-dog CYP differences. Canine CYP1A2, which metabolizes phenacetin, caffeine, and theophylline, is the most widely studied polymorphic canine CYP. A single nucleotide polymorphism resulting in a CYP1A2 premature stop codon (c.1117C>T; R383X) with a complete lack of enzyme is highly prevalent in certain dog breeds including Beagle and Irish wolfhound. This polymorphism was shown to substantially affect the pharmacokinetics of several experimental compounds in Beagles during preclinical drug development. However, the impact on the pharmacokinetics of phenacetin (a substrate specific for human CYP1A2) was quite modest probably because other canine CYPs are capable of metabolizing phenacetin. Other canine CYPs with known genetic polymorphisms include CYP2C41 (gene deletion), as well as CYP2D15, CYP2E1, and CYP3A12 (coding SNPs). However the impact of these variants on drug metabolism in vitro or on drug pharmacokinetics is unknown. Future systematic investigations are needed to comprehensively identify CYP genetic polymorphisms that are predictive of drug effects in canine patients.

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Section: Dog-human Cyp Similarities and Differencesmentioning

confidence: 99%

Canine Cytochrome P-450 Pharmacogenetics

Court

2013

Veterinary Clinics of North America: Small Animal Practice

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“…There is a great interest in quantifying endogenous human drug metabolizing enzymes and drug transporters to assist better understanding of the absorption, distribution, metabolism, and excretion (ADME) of drugs. LC-MS/MS-based quantitation of the cytochrome P450 enzyme family (Seibert et al, 2009;Sakamoto et al, 2011;Williamson et al, 2011;Heikkinen et al, 2012), transporters (Li et al, 2008;Sakamoto et al, 2011), and UDP-glucuronosyltransferases (Sakamoto et al, 2011) have been reported in the literature to assist the development of predictive clearance models. In this report, we described a fast, sensitive, and robust LC-MS/MS method to quantify absolute AOX1 levels and the method was applied to AOX1 quantification of human liver cytosol.…”

Section: Introductionmentioning

confidence: 99%

Aldehyde Oxidase 1 (AOX1) in Human Liver Cytosols: Quantitative Characterization of AOX1 Expression Level and Activity Relationship

Han

et al. 2013

Drug Metab Dispos

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Aldehyde oxidase 1 (AOX1) is a cytosolic enzyme highly expressed in liver and plays a key role in metabolizing drugs containing aromatic azaheterocyclic substituents. Rapid metabolism catalyzed by AOX1 can cause a drug to exhibit high clearance, low exposure, and hence decreased efficacy or even increased toxicity (if AOX1 generated metabolites are toxic). There is a need to develop the correlation between AOX1 expression levels and AOX1-substrate clearance. A fast, sensitive, and robust liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed to quantify AOX1 in human liver cytosol for the first time. This LC-MS/MS method includes a straightforward ultrafiltration fractionation step and gives great selectivity and wide dynamic range (5.2 pM to 20.7 nM). The AOX1 levels in human liver cytosols of 20 donors were quantified using this method to investigate individual differences in AOX1 expression. No significant individual or gender differences in AOX1 levels were observed, although male donors exhibited a broader distribution than female donors (0.74-2.30 pmol/mg versus 0.74-1.69 pmol/mg, respectively). The AOX1 protein levels measured by LC-MS/MS were consistent with those measured by an enzyme-linked immunosorbent assay. Several donors have a normal AOX1 protein level but low enzyme activity, which might be due to cofactor deficiency, single nucleotide polymorphism, or homodimer dissociation. Cytosols from donors with chronic alcohol consumption had low AOX1-catalyzed carbazeran oxidation activities (<51 ml/min per milligram compared with a median of 455 ml/min per milligram), but preserved similar AOX1 protein expression levels (approximately 15% less than the median value)

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“…Linear correlations demonstrated between metabolism or transport of specific probes and corresponding protein abundance in vitro confirm that protein concentration is a determinant of activity. However, it is well recognized that enzyme and transporter activity is also affected by other factors such as accessory proteins, energy sources, co‐factors, and the specific membrane and aqueous environments of the protein .…”

Section: Examples Of the Utility Of Protein Abundance Data For Ivive mentioning

confidence: 64%

“…Hepatic elimination is a significant determinant of PK and it is not surprising that the majority of LC‐MS/MS‐based proteomic studies characterizing enzyme and transporter abundances have done so in the human liver [, . Furthermore, other human tissues important for PK, namely intestine , kidneys , and lungs , and the blood–brain barrier have received attention as well as various tissues in animal species . A significant part of the existing data on CYP abundance is based on immunochemical protein quantification methods and consequently applications for IVIVE and PBPK have employed these data.…”

Section: Examples Of the Utility Of Protein Abundance Data For Ivive mentioning

confidence: 99%

“…However, if this is to be achieved then certain recommendations need to be followed to enable pooling of data from various sources: Losses of material during sample preparation should be quantified and reported thoroughly. This has been a common practice in quantification of microsomal metabolic enzymes , but generally has not been done in transporter quantification studies . Methodology applied must be reported transparently in every detail. Quantification results in individual samples should be published as data supplements. Sample processing should be replicated in order to enable separation of technical and biological variability. A set of tissue samples should be made available to allow for direct comparison and analysis of methodological differences between laboratories and enabling normalization of results between studies. …”

Section: The Future: What Is Needed and Where Can We Expect To See Prmentioning

confidence: 99%

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The role of quantitative ADME proteomics to support construction of physiologically based pharmacokinetic models for use in small molecule drug development

Heikkinen

Lignet

Cutler

et al. 2015

Proteomics Clinical Apps

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Pharmacokinetics (PK) refers to the time course of drug concentrations in the body and since knowledge of PK aids understanding of drug efficacy and safety, numerous PK studies are performed in animals and humans during the drug development process. In vitro to in vivo extrapolation and physiologically based pharmacokinetic (PBPK) modeling are tools that integrate data from various in silico, in vitro, and in vivo sources to deliver mechanistic quantitative simulations of in vivo PK. PBPK models are used to predict human PK and to evaluate the effects of intrinsic factors such as organ dysfunction, age, and genetics as well as extrinsic factors such as co-administered drugs. In recent years, the use of PBPK within the industry has greatly increased. However, insufficient data on how the abundance of metabolic enzymes and membrane transporters vary in different human patient populations and in different species has been a limitation. A major advance is therefore expected through reliable quantification of the abundance of these proteins in tissues. This review describes the role of PBPK modeling in drug discovery and development, outlines the assumptions involved in integrating protein abundance data, and describes the advances made and expected in determining abundance of relevant proteins through mass spectrometric techniques.

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Mass Spectrometry-Based Quantification of CYP Enzymes to Establish In Vitro/In Vivo Scaling Factors for Intestinal and Hepatic Metabolism in Beagle Dog

Abstract: CYP expression in dog liver and intestine was characterized, providing a basis for in vitro/in vivo scaling of intestinal and hepatic metabolism.

Cited by 29 publications

References 32 publications

Canine Cytochrome P-450 Pharmacogenetics

Canine Cytochrome P-450 Pharmacogenetics

Aldehyde Oxidase 1 (AOX1) in Human Liver Cytosols: Quantitative Characterization of AOX1 Expression Level and Activity Relationship

The role of quantitative ADME proteomics to support construction of physiologically based pharmacokinetic models for use in small molecule drug development

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