J. Steven Leeder

Cytochrome P450 3A: ontogeny and drug disposition.

The maturation of organ systems during fetal life and childhood exerts a profound effect on drug disposition. The maturation of drug-metabolising enzymes is probably the predominant factor accounting for age-associated changes in non-renal drug clearance. The group of drug-metabolising enzymes most studied are the cytochrome P450 (CYP) superfamily. The CYP3A subfamily is the most abundant group of CYP enzymes in the liver and consists of at least 3 isoforms: CYP3A4, 3A5 and 3A7. Many drugs are mainly metabolised by the CYP3A subfamily. Therefore, maturational changes in CYP3A ontogeny may impact on the clinical pharmacokinetics of these drugs.CYP3A4 is the most abundantly expressed CYP and accounts for approximately 30 to 40% of the total CYP content in human adult liver and small intestine. CYP3A5 is 83% homologous to CYP3A4, is expressed at a much lower level than CYP3A4 in the liver, but is the main CYP3A isoform in the kidney. CYP3A7 is the major CYP isoform detected in human embryonic, fetal and newborn liver, but is also detected in adult liver, although at a much lower level than CYP3A4. Substrate specificity for the individual isoforms has not been fully elucidated. Because of large interindividual differences in CYP3A4 and 3A5 expression and activity, genetic polymorphisms have been suggested. However, although some gene mutations have been identified, the impact of these mutations on the pharmacokinetics of CYP3A substrates has to be established.Ontogeny of CYP3A activity has been studied in vitro and in vivo. CYP3A7 activity is high during embryonic and fetal life and decreases rapidly during the first week of life. Conversely, CYP3A4 is very low before birth but increases rapidly thereafter, reaching 50% of adult levels between 6 and 12 months of age. During infancy, CYP3A4 activity appears to be slightly higher than that of adults. Large interindividual variations in CYP3A5 expression and activity were observed during all stages of development, but no apparent developmental pattern of CYP3A5 activity has been identified to date.Profound changes occur in the activity of CYP3A isoforms during all stages of development. These changes have, in many instances, proven to be of clinical significance when treatment involves drugs that are substrates, inhibitors or inducers of CYP3A. Investigators and clinicians should consider the impact of ontogeny on CYP3A in both pharmacokinetic study design and data interpretation, as well as when prescribing drugs to children.

Glucuronidation in Humans Pharmacogenetic and Developmental Aspects

During human development impressive changes in drug disposition occur. An important determinant of drug clearance is metabolism, something that is not only determined by ontogenic regulation but also by genetic processes which add to the variability of drug metabolism during different stages of childhood. Therefore, an understanding of the developmental regulation of different metabolic pathways, together with information on the genetic determinants of drug metabolism, will increase the knowledge of inter- and intraindividual variability in drug disposition during childhood.Conjugation has historically received less attention than cytochrome P450 metabolism. An important group of conjugation reactions are catalysed by the uridine 5′-diphosphate (UDP)-glucuronosyltransferases (UGTs); to date at least 10 different UGT isoforms have been identified. The UGTs are not only involved in the metabolism of many drugs [e.g. morphine, paracetamol (acetaminophen)] but also capable of the biotransformation of important endogenous substrates (e.g. bilirubin, ethinylestradiol) and several xenobiotics. Isoform specificity for these substrates has, however, not been fully characterised.Serious adverse events associated with chloramphenicol toxicity in the neonate have highlighted the importance of developmental changes in UGT activity. However, isoform-specific differences preclude the generalisation of a simple developmental pattern for UGT activity. UGT2B7 is the only UGT isoform for which ontogeny has been characterised both in vitro and in vivo, using morphine as the probe drug. However, no general developmental pattern for the individual UGT isoforms which might be of value for the clinician is currently available.Genetic polymorphisms have been identified for the UGT family. Not only for the UGT1A gene, which reduces bilirubin glucuronidation, leading to genetic hyperbilirubinaemia (the Crigler-Najjar and Gilbert’s syndromes), but also for 3 other UGT isoforms. However, the impact of these genetic differences on drug metabolism remains to be established because of overlapping isoform specificity of the drugs studied, as well as a lack of specific probe substrates to test the activity of individual UGT isoforms in relation to these gene mutations.Clearly, an information gap exists regarding the developmental and genetic aspects of UGT regulation and its potential impact on therapy. More research is needed on the pharmacogenetics and ontogeny of the UGTs for effective translation of scientific information into clinically applicable knowledge.

Systematic genetic and genomic analysis of cytochrome P450 enzyme activities in human liver

Liver cytochrome P450s (P450s) play critical roles in drug metabolism, toxicology, and metabolic processes. Despite rapid progress in the understanding of these enzymes, a systematic investigation of the full spectrum of functionality of individual P450s, the interrelationship or networks connecting them, and the genetic control of each gene/enzyme is lacking. To this end, we genotyped, expression-profiled, and measured P450 activities of 466 human liver samples and applied a systems biology approach via the integration of genetics, gene expression, and enzyme activity measurements. We found that most P450s were positively correlated among themselves and were highly correlated with known regulators as well as thousands of other genes enriched for pathways relevant to the metabolism of drugs, fatty acids, amino acids, and steroids. Genome-wide association analyses between genetic polymorphisms and P450 expression or enzyme activities revealed sets of SNPs associated with P450 traits, and suggested the existence of both cis-regulation of P450 expression (especially for CYP2D6) and more complex trans-regulation of P450 activity. Several novel SNPs associated with CYP2D6 expression and enzyme activity were validated in an independent human cohort. By constructing a weighted coexpression network and a Bayesian regulatory network, we defined the human liver transcriptional network structure, uncovered subnetworks representative of the P450 regulatory system, and identified novel candidate regulatory genes, namely, EHHADH, SLC10A1, and AKR1D1. The P450 subnetworks were then validated using gene signatures responsive to ligands of known P450 regulators in mouse and rat. This systematic survey provides a comprehensive view of the functionality, genetic control, and interactions of P450s.

Mechanisms of Idiosyncratic Hypersensitivity Reactions to Antiepileptic Drugs

Summary: Hypersensitivity reactions to the aromatic antiepileptic drugs (AEDs) phenytoin (PHT) and carbamazepine (CBZ) appear to have an immune etiology. Current models of drug hypersensitivity center around the concept of drug bioactivation to reactive metabolites that irreversibly modify cellular proteins. These modified proteins are believed to initiate (or serve as targets of) an autoimmune‐like attack on specific drug‐modified proteins in target organs (e.g., liver, skin) of susceptible individuals. Consistent with this model, antibodies to drug‐modified and native proteins have been identified in the sera of patients experiencing several drug hypersensitivity reactions. New models must incorporate an understanding of the mechanisms by which drug‐modified proteins are processed and presented to the immune system in the appropriate context to culminate in the clinical manifestations of “hypersensitivity.” Idiosyncratic toxicities associated with new AEDs, such as lamotrigine and felbamate, appear mechanistically distinct from PHT and CBZ hypersensitivity but may involve similar processes: bioactivation, detoxification, covalent adduct formation, processing and presentation of antigen to the immune system, and consequent formation of antibody and T‐cell immune effectors. The goal of research is to develop a “susceptibility profile” for identifying individuals at risk for these forms of drug toxicity.

Pharmacokinetics and CYP2D6 genotypes do not predict metoprolol adverse events or efficacy in hypertension

β‐Blocker use can be associated with adverse effects that may have an impact on adherence or harm patients. The commonly prescribed β‐blocker metoprolol is metabolized by the polymorphic cytochrome P450 (CYP) 2D6 enzyme, resulting in widely variable drug exposure. We investigated whether metoprolol plasma concentrations, CYP2D6 polymorphisms, or genotype‐derived phenotype was associated with adverse effects or efficacy in patients with hypertension.

Combined phenotypic assessment of CYP1A2, CYP2C19, CYP2D6, CYP3A, N‐acetyltransferase‐2, and xanthine oxidase with the “Cooperstown cocktail”

Simultaneous administration of several probes enhances the utility of phenotyping, but poor specificity, side effects, and use of drugs not approved by the Food and Drug Administration limit the usefulness of prior phenotyping cocktails.

Combined phenotypic assessment of cytochrome p450 1A2, 2C9, 2C19, 2D6, and 3A, N-acetyltransferase-2, and xanthine oxidase activities with the "Cooperstown 5+1 cocktail".

Previously, we have validated a 4‐drug phenotyping cocktail, the “Cooperstown cocktail,” using caffeine (cytochrome P450 [CYP] 1A2, N‐acetyltransferase‐2 [NAT2], and xanthine oxidase [XO]), dextromethorphan (CYP2D6), omeprazole (CYP2C19), and intravenous midazolam (hepatic CYP3A). Data suggest that warfarin can be used as a safe and accurate biomarker for CYP2C9, and if warfarin is administered with vitamin K, the pharmacodynamic effect is ablated. Twelve subjects received the Cooperstown cocktail, warfarin plus vitamin K, and both sets of biomarkers (Cooperstown 5+1 cocktail) in a randomized crossover fashion. On the basis of log‐transformed data and a paired t test, no significant difference was seen for S‐warfarin area under the serum concentration–time curve from time 0 to infinity (P = .09), omeprazole metabolic ratio (P = .374), caffeine metabolic ratio (P = .169 for CYP1A2 activity), midazolam plasma clearance (P = .573), or dextromethorphan metabolic ratio (P = .747) with the Cooperstown cocktail, warfarin plus vitamin K alone, or the Cooperstown 5+1 cocktail. During drug administration, the only side effect was mild and short‐lived sedation after intravenous midazolam administration. Phenotypic measurements were in concordance with the subjects CYP2C9, CYP2C19, and CYP2D6 genotypes. The Cooperstown 5+1 cocktail may be used to simultaneously assess the activities of CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A, NAT2, and XO.

Cytochrome P4502D6 (CYP2D6) Gene Locus Heterogeneity: Characterization of Gene Duplication Events

Duplications and multiplications of active CYP2D6 genes can cause ultrarapid drug metabolism and lead to therapeutic failure. Multiple functional and non‐functional duplication alleles have been further characterized. Duplications were detected by long‐range polymerase chain reaction (PCR), PCR‐restriction fragment length polymorphism, and sequence analysis. A PCR fragment encompassing the entire duplicated gene was utilized for detailed characterization. Duplications occurred at 1.3, 5.75, and 2.0% in Caucasian, African American, and racially mixed populations, respectively (n=887 total). Of those 28, 47, and 17% were non‐functional CYP2D6*4 × N. Twelve unique duplication alleles were detected: *1 × N, *2 × N, *4 × N, *6 × N, *10 × N, *17 × N, *17 × N[spacer], *29 × N, *35 × N, *43 × N, *45 × N, and a novel non‐functional tandem arrangement of a chimeric 2D7/2D6 and *1 gene. All novel duplications except *35 × N were found in African Americans. Accurate identification of gene duplication events is essential to avoid false‐positive ultrarapid metabolism assignments and thus, overestimation of predicted activity and increased risk for unwanted adverse events.

A Method for Meta-Analysis of Epidemiological Studies

This article presents a stepwise approach for conducting a meta-analysis of epidemiological studies based on proposed guidelines. This systematic method is recommended for practitioners evaluating epidemiological studies in the literature to arrive at an overall quantitative estimate of the impact of a treatment. Bendectin is used as an illustrative example. Meta-analysts should establish a priori the purpose of the analysis and a complete protocol. This protocol should be adhered to, and all steps performed should be recorded in detail. To aid in developing such a protocol, we present methods the researcher can use to perform each of 22 steps in six major areas. The illustrative meta-analysis confirmed previous traditional narrative literature reviews that Bendectin is not related to teratogenic outcomes in humans. The overall summary odds ratio was 1.01 (χ2 = 0.05, p = 0.815) with a 95 percent confidence interval of 0.66–1.55. When the studies were separated according to study type, the summary odds ratio for cohort studies was 0.95 with a 95 percent confidence interval of 0.62–1.45. For case-control studies, the summary odds ratio was 1.27 with a 95 percent confidence interval of 0.83–1.94. The corresponding chi-square values were not statistically significant at the p = 0.05 level.

Quantitation of three‐month intraindividual variability and influence of sex and menstrual cycle phase on CYP1A2, N‐acetyltransferase‐2, and xanthine oxidase activity determined with caffeine phenotyping

To evaluate intraindividual variability and the effects of sex and menstrual cycle phase on the activity of cytochrome P450 1A2 (CYP1A2), N‐acetyltransferase 2 (NAT2), and xanthine oxidase.